Method of reducing the risk of bone fracture

ABSTRACT

The invention relates to a method for increasing the toughness and/or stiffness of bone and/or reducing the likelihood and/or severity of bone fracture by administering a parathyroid hormone. The method can be employed to increase toughness or stiffness of bone at a site of a potential or actual trauma, such as the hip or spine of a person at risk of or suffering from osteoporosis. The method of the invention can reduce the incidence of vertebral fractures, reduce the incidence of multiple vertebral fractures, reduce the severity of vertebral fracture, and/or reduce the incidence of non-vertebral fracture.

TECHNICAL FIELD

This invention relates to methods for increasing the toughness and/orstiffness of bone and/or reducing the likelihood and/or severity of bonefracture by administering a parathyroid hormone. More particularly, theinvention relates a method for increasing toughness or stiffness of boneat a site of a potential or actual trauma, such as the hip or spine of aperson at risk of or suffering from osteoporosis. More particularly, theinvention relates to a method of reducing the incidence of vertebralfracture, reducing the incidence of multiple vertebral fractures,reducing the severity of vertebral fracture, and/or reducing theincidence of non-vertebral fracture.

BACKGROUND OF THE INVENTION

Existing agents such as estrogen, bisphosphonates, fluoride, orcalcitonin can prevent bone loss and induce a 3-5% increase of bone massby refilling the remodeling space, but net bone formation is notsignificantly stimulated. The retention of bone by inhibition of boneturnover may not be sufficient protection against fracture risk forpatients who already have significant bone loss. Anabolic agents thatincrease bone strength by stimulating bone formation preferentially mayprovide better protection against fracture in patients with establishedosteoporosis.

Parathyroid hormone (PTH) is a secreted, 84 amino acid product of themammalian parathyroid gland that controls serum calcium levels throughits action on various tissues, including bone. The N-terminal 34 aminoacids of bovine and human PTH (PTH(1-34)) is deemed biologicallyequivalent to the full length hormone. Other amino terminal fragments ofPTH (including 1-31 and 1-38 for example), or PTHrP (PTH-relatedpeptide/protein) or analogues of either or both, that activate thePTH/PTHrP receptor (PTHI receptor) have shown similar biologic effectson bone mass, although the magnitude of such effects may vary.

Studies in humans with various forms of PTH have demonstrated ananabolic effect on bone, and have prompted significant interest in itsuse for the treatment of osteoporosis and related bone disorders. Thesignificant anabolic effects of PTH on bone, including stimulation ofbonc formation which results in a net gain in bone mass and/or strength,have been demonstrated in many animal models and in humans.

It is commonly believed that PTH administration in humans and inrelevant animal models has a negative effect on cortical bone. In fact,naturally occurring increases in endogenous PTH, which occur in thedisorder hyperparathyroidism, result in thinning of cortical boneaccompanied by an increase in connectivity and mass of trabecular bone.Past studies suggest that when Haversian cortical bone (found in humansand higher mammals) remodels under the influence of PTH, there will be are-distribution of bone such that cortical bone mass and strengthdecrease, while trabecular bone increases in mass and strength. Forexample, in published clinical studies of administering PTH, corticalbone mass decreased after treatment with exogenous PTH and thesefindings have raised concern that the treatment of PTH will lead toreduced cortical bone mass and strength. One concern raised by suchstudies is that there would be a loss of total skeletal bone mass due tothe loss of cortical bone. This is of high clinical relevance as, inosteoporosis, the greater loss of trabecular bone compared to loss ofcortical bone, means that mechanical loading is predominantly borne bythe remaining cortical bone. Continued loss of cortical bone wouldincrease the fracture risk. Therefore, it is important that atherapeutic agent for osteoporosis maintain or increase a subjectsresidual cortical bone.

The effects of PTH on cortical bone have been investigated in nonhumananimals with Haversian remodeling, such as dogs, ferrets, sheep andmonkeys, but sample sizes are typically too small for reliablestatistical analysis. The impact of the changes induced by PTH treatmenton mechanical properties of cortical bone in such animals remainsunknown. Published studies of rodents have shown increased cortical bonemass during administration of PTH but a loss of this benefit afterwithdrawal of PTH. However, rodent cortical bone has a distinctlydifferent structure from Haversian cortical bone, and remodels bysurface appositional formation and resorption, rather than byintracortical remodeling of osteons. Furthermore, technologicallimitations in biomechanical testing on the relatively short bones ofrodents give rise to artifacts of measurement when an agent, such as aPTH, alters bone geometry to thicken the bone. Such artifacts makeextrapolation of rat cortical bone responses to those of humans or otheranimals with osteonal remodeling unreliable. Therefore, the existingdata for animals, like humans, undergoing Haversian remodeling indicatesthat PTH may have an adverse impact on cortical bone, causing net lossof bone mass through depletion of cortical bone.

As a consequence, it has been a popular belief regarding the action ofPTH that patients require concurrent or subsequent treatment with anantiresorptive to minimize loss of bone induced by PTH. In fact, thismodel has been the basis for several clinical studies in women. Forexample, three clinical studies have used PTH in post-menopausal womenon concurrent therapy with calcitonin or estrogen, or in premenopausalwomen taking GnRH agonist, Synarel, for endometriosis. The opposingeffects of estrogen and PTH on cortical bone turnover make itparticularly difficult to observe effects of just PTH during combinationtherapy with these two agents.

There remains a need for a method for employing a PTH to increasestrength and stiffness of bone in humans and other animals exhibitingHaversian remodeling, and for reducing the incidence of fracture ofbones in these animals. Furthermore, there remains a need for a methodfor increasing the quality and amount of cortical bone.

SUMMARY OF THE INVENTION

The present invention includes a method for increasing the toughnessand/or stiffness of bone, preferably cortical bone, and/or reducing theincidence and/or severity of fracture by administering a parathyroidhormone. More particularly, the invention relates to a method forincreasing toughness or stiffness of bone at a site of a potential oractual trauma. Increasing toughness and/or stiffness of bone can bemanifested in numerous ways known to those of skill in the art, such asincreasing bone mineral density, increasing bone mineral content,increasing work to failure, and the like. In one embodiment, the methodof the invention reduces the incidence or severity of vertebral and/ornon-vertebral fractures. The method of the invention can be used todecrease the risk of such fractures or for treating such fractures. Inparticular, the method of the invention can reduce the incidence ofvertebral and/or non-vertebral fracture, reduce the severity ofvertebral fracture, reduce the incidence of multiple vertebral fracture,improve bone quality, and the like.

The method can increase toughness or stiffness at a site of a potentialtrauma, such as a hip or spine of a person with osteoporosis, or atanother site having abnormally low bone mass or poor bone structure. Themethod can also increase bone toughness or stiffness at a site of anactual trauma, such as a fracture, for example, in a hip or vertebra. Apreferred subject for the method of the invention is a woman or man atrisk for or having osteoporosis, preferably a postmenopausal woman, andis independent of concurrent hormone replacement therapy (HRT), estrogenor equivalent therapy, or antiresorptive therapy. In one embodiment, thepatient also receives supplements of calcium and/or vitamin D.

A parathyroid hormone, such as the N-terminal amino acids 1-34 ofrecombinant human parathyroid hormone, can be administered eithercyclically or intermittently. Preferably, cyclic administration includesadministering PTH for 2 or more remodeling cycles and withdrawing PTHfor one or more remodeling cycles. Further, according to the method ofthe invention, the increases in toughness and/or stiffness of a bone canpersist for several remodeling cycles, or up to several years, after thelast administration of a parathyroid hormone.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show that BMD (bone mineral density) and BMC (bonemineral content) in the femoral midshaft (cortical bone) (A) and in theproximal femur (cancellous bone +cortical bone) (B) were significantlygreater in PTH-treated animals than controls at both doses.

FIGS. 2A through 2D show the effects of PTH on mechanical strength andcross sectional moment of inertia (CSMI) in the cortical bone of thefemoral midshaft.

FIG. 3 illustrates the percent change in DXA measures of whole bonemineral content in control and treatment groups.

FIGS. 4A-C illustrate the percent change in DXA measures of the spinefor control and treatment groups in the lumbar vertebrae 2-4 for bonearea (A), bone mineral content (B), and bone mineral density (C).

FIGS. 5A and 5B illustrate the increase in bone mass (A) and bonestrength (B) in lumbar vertebrae of primates treated with a parathyroidhormone.

FIGS. 6A and 6B illustrate the increase in strength of femur neck (A)and the constant strength of humerus mid-diaphysis (B) in primatestreated with a parathyroid hormone.

FIG. 7 illustrates activation of bone formation rates on endosteal andperiosteal surfaces of the midshaft humerus.

FIG. 8 illustrates the histogram analysis of the shift in bone voxeldensities in lumbar vertebra, resulting from PTH treatment compared tocontrol. Note the increase in density in cortical bone compartment afterwithdrawal of PTH treatment.

FIG. 9 illustrates increases in lumbar spine BMD through 23 months oftreatment of patients with either 20 μg/kg/day PTH or 40 μg/kg/day PTH,compared to placebo treated controls.

FIG. 10 illustrates increases in femur and hip neck BMD through 24months of treatment of patients with either 20 μg/kg/day PTH or 40μg/kg/day PTH, compared to placebo treated controls.

DETAILED DESCRIPTION

The invention relates to a method for increasing bone toughness and/orstiffness, and/or reducing incidence of fracture in a subject byadministering a parathyroid hormone. The method can be employed toincrease stiffness and/or toughness at a site of a potential trauma orat a site of an actual trauma. Trauma generally includes fracture,surgical trauma, joint replacement, orthopedic procedures, and the like.Increasing bone toughness and/or stiffness generally includes increasingmineral density of cortical bone, increasing strength of bone,increasing resistance to loading, and the like. Reducing incidence offracture generally includes reducing the likelihood or actual incidenceof fracture for a subject compared to an untreated control population.

As used herein, ultimate force refers to maximum force that a bonespecimen sustains; stiffness refers to the slope of the linear portionof a load-deformation curve; and work to failure refers to the areaunder the load-deformation curve before failure. Each of these can bemeasured and calculated by methods standard in the bone study art. Theseparameters are structural properties that depend on intrinsic materialproperties and geometry, and can be determined as described in TurnerCH, Burr DB 1993 “Basic biomechanical measurements of bone: a tutorial.”Bone 14:595-608, which is incorporated herein by reference. Ultimateforce, stiffness, and work to failure can be normalized to obtainintrinsic material properties such as ultimate stress, elastic modulus,and toughness, which are independent of size and shape. As used herein,ultimate stress refers to maximum stress that a specimen can sustain;elastic modulus refers to material intrinsic stiffness; and toughnessrefers to resistance to fracture per unit volume. Each of these can bedetermined by methods known in the art. Id. Femoral bone strength, asreferred to herein, can be measured at the femur neck, or at themidshaft typically using three-point or four-point bending at the lattersite.

Bone Trauma

The method of the invention is of benefit to a subject that may sufferor have suffered trauma to one or more bones. The method can benefitmammalian subjects, such as humans, horses, dogs, and cats, inparticular, humans. Bone trauma can be a problem for racing horses anddogs, and also for household pets. A human can suffer any of a varietyof bone traumas due, for example, to accident, medical intervention,disease, or disorder. In the young, bone trauma is likely due tofracture, medical intervention to repair a fracture, or the repair ofjoints or connective tissue damaged, for example, through athletics.Other types of bone trauma, such as those from osteoporosis,degenerative bone disease (such as arthritis or osteoarthritis), hipreplacement, or secondary conditions associated with therapy for othersystemic conditions (e.g., glucocorticoid osteoporosis, burns or organtransplantation) are found most often in older people.

Preferred subjects include a human, preferably a woman, at risk for orsuffering from osteoporosis. Risk factors for osteoporosis are known inthe art and include hypogonadal conditions in men and women,irrespective of age, conditions, diseases or drugs that inducehypogonadism, nutritional factors associated with osteoporosis (lowcalcium or vitamin D being the most common), smoking, alcohol, drugsassociated with bone loss (such as glucocorticoids, thyroxine, heparin,lithium, anticonvulsants etc.), loss of eyesight that predisposes tofalls, space travel, immobilization, chronic hospitalization or bedrest, and other systemic diseases that have been linked to increasedrisk of osteoporosis. Indications of the presence of osteoporosis areknown in the art and include radiological evidence of at least onevertebral compression fracture, low bone mass (typically at least Istandard deviation below mean young normal values), and/or atraumaticfractures.

Osteoporosis can lead, for example, to vertebral and/or non-vertebralfractures. Examples of non-vertebral fractures include a hip fracture, afracture of a distal forearm, a fracture of a proximal humerus, afracture of a wrist, a fracture of a radius, a fracture of an ankle, afracture of an humerus, a fracture of a rib, a fracture of a foot, afracture of a pelvis, or a combination of these. The method of theinvention can be used to decrease the risk of such fractures or fortreating such fractures. The risk of fracture is diminished and thehealing of a fracture is aided by increasing the strength and/orstiffness of bone, for example, in the hip, the spine or both. A typicalwoman at risk for osteoporosis is a postmenopausal woman or apremenopausal, hypogonadal woman. A preferred subject is apostmenopausal woman, and is independent of concurrent hormonereplacement therapy (HRT), estrogen or equivalent therapy, orantiresorptive therapy. The method of invention can benefit a subject atany stage of osteoporosis, but especially in the early and advancedstages.

The present invention provides a method, in particular, effective toprevent or reduce the incidence of fractures in a subject with or atrisk of progressing to osteoporosis. For example, the present inventioncan reduce the incidence of vertebral and/or non-vertebral fracture,reduce the severity of vertebral fracture, reduce the incidence ofmultiple vertebral fracture, improve bone quality, and the like. Inanother embodiment, the method of the present invention can benefitpatients with low bone mass or prior fracture who are at risk for futuremultiple skeletal fractures, such as patients in which spinalosteoporosis may be progressing rapidly.

Other subjects can also be at risk of or suffer bone trauma and canbenefit from the method of the invention. For example, a wide variety ofsubjects at risk of one or more of the fractures identified above, cananticipate surgery resulting in bone trauma, or may undergo anorthopedic procedure that manipulates a bone at a skeletal site ofabnormally low bone mass or poor bone structure, or deficient inmineral. For example, recovery of function after a surgery such as ajoint replacement (e.g. knee or hip) or spine bracing, or otherprocedures that immobilize a bone or skeleton can improve due to themethod of the invention. The method of the invention can also aidrecovery from orthopedic procedures that manipulate a bone at a site ofabnormally low bone mass or poor bone structure, which proceduresinclude surgical division of bone, including osteotomies, jointreplacement where loss of bone structure requires restructuring withacetabulum shelf creation and prevention of prosthesis drift, forexample. Other suitable subjects for practice of the present inventioninclude those suffering from hypoparathyroidism or kyphosis, who canundergo trauma related to, or caused by, hypoparathyroidism orprogression of kyphosis.

Bone Toughness and Stiffness

The method of the invention reduces the risk of trauma or aids recoveryfrom trauma by increasing bone toughness, stiffness or both. Generallytoughness or stiffness of bone results from mass and strength ofcortical, trabecular, and cancerous bone. The method of the inventioncan provide levels of bone toughness, stiffness, mass, and/or strengthwithin or above the range of the normal population. Preferably theinvention provides increased levels relative to the levels resultingfrom trauma or giving rise to risk of trauma. Increasing toughness,stiffness, or both decreases risk or probability of fracture compared toan untreated control population.

Certain characteristics of bone when increased provide increased bonetoughness and/or stiffness. Such characteristics include bone mineraldensity (BMD), bone mineral content (BMC), activation frequency or boneformation rate, trabecular number, trabecular thickness, trabecular andother connectivity, periosteal and endocortical bone formation, corticalporosity, cross sectional bone area and bone mass, resistance toloading, and/or work to failure. An increase in one or more of thesecharacteristics is a preferred outcome of the method of the invention.

Certain characteristics of bone, such as marrow space and elasticmodulus when decreased provide increased toughness and/or stiffness ofbone. Younger (tougher and stiffer) bone has crystallites that aregenerally smaller than crystallites of older bone. Thus, generallyreducing the size of bone crystallites increases toughness and stiffnessof bone, and can reduce incidence of fracture. In addition, maturing thecrystallites of a bone can provide additional desirable characteristicsto the bone, including increased toughness and stiffness of bone and/orcan reduced incidence of fracture. A decrease in one or more of thesecharacteristics can be a preferred outcome of the method of theinvention.

The method of the invention is effective for increasing the toughnessand/or stiffness of any of several bones. For example, the presentmethod can increase the toughness and/or stiffness of bones including ahip bone, such as an ilium, a leg bone, such as a femur, a bone from thespine, such as a vertebra, or a bone from an arm, such as a,distalforearm bone or a proximal humerus. This increase in toughness and/orstiffness can be found throughout the bone, or localized to certainportions of the bone. For example, toughness and/or stiffness of a femurcan be increased by increasing the toughness and/or stiffness of a femurneck or a femur trochantera. Toughness and/or stiffness of a hip can beincreased by increasing the toughness and/or stiffness of an iliac crestor iliac spine. Toughness and/or stiffness of a vertebra can beincreased by increasing the toughness and/or stiffness of a pedicle,lamina, or body. Advantageously, the effect is on vertebra in certainportions of the spine, such as cervical, thoracic, lumbar, sacral,and/or coccygeal vertebrae. Preferably the effect is on one or moremid-thoracic and/or upper lumbar vertebrae.

The increase in toughness and/or stiffness can be found in each of thetypes of bone, or predominantly in one type of the bone. Types of boneinclude spongy (cancellous, trabecular, or lamellar) bone and compact(cortical or dense) bone and the fracture callus. The method of theinvention preferably increases toughness and/or stiffness through itseffects on cancellous and cortical bone, or on cortical bone alone.Trabecular bone, bone to which connective tissue is attached can also betoughened and/or stiffened by the present method. For example, it isadvantageous to provide additional toughness at a site of attachment fora ligament, a tendon, and/or a muscle.

In another aspect of the invention, increasing toughness or stiffnesscan reduce incidence of fracture. In this aspect, increasing toughnessor stiffness can include reducing incidence of vertebral fracture,reducing incidence of severe fracture, reducing incidence of moderatefracture, reducing incidence of non-vertebral fracture, reducingincidence of multiple fracture, or a combination thereof.

Parathyroid Hormone

As active ingredient, the composition or solution may incorporate thefull length, 84 amino acid form of parathyroid hormone, particularly thehuman form, hPTH (1-84), obtained either recombinantly, by peptidesynthesis or by extraction from human fluid. See, for example, U.S. Pat.No. 5,208,041, incorporated herein by reference. The amino acid sequencefor hPTH (1-84) is reported by Kimura et al. in Biochem. Biophys. Res.Comm., 114(2):493.

The composition or solution may also incorporate as active ingredientfragments or variants of fragments of human PTH or of rat, porcine orbovine PTH that have human PTH activity as determined in theovariectomized rat model of osteoporosis reported by Kimmel et al.,Endocrinology, 1993, 32(4):1577.

The parathyroid hormone fragments desirably incorporate at least thefirst 28 N-terminal residues, such as PTH(1-28), PTH(1-31), PTH(1-34),PTH(1-37), PTH(1-38) and PTH(1-41). Alternatives in the form of PTHvariants incorporate from 1 to 5 amino acid substitutions that improvePTH stability and half-life, such as the replacement of methionineresidues at positions 8 and/or 18 with leucine or other hydrophobicamino acid that improves PTH stability against oxidation and thereplacement of amino acids in the 25-27 region with trypsin-insensitiveamino acids such as histidine or other amino acid that improves PTHstability against protease. Other suitable forms of PTH include PTHrP,PTHrP(1-34), PTHrP(1-36) and analogs of PTH or PTHrP that activate thePTH1 receptor. These forms of PTH are embraced by the term “parathyroidhormone” as used generically herein. The hormones may be obtained byknown recombinant or synthetic methods, such as described in U.S. Pat.Nos. 4,086,196 and 5,556,940, incorporated herein by reference.

The preferred hormone is human PTH(1-34), also known as teriparatide.Stabilized solutions of human PTH(1-34), such as recombinant humanPTH(1-34) (rhPTH(1-34), that can be employed in the present method aredescribed in U.S. patent application Ser. No. 60/069,075, incorporatedherein by reference. Crystalline forms of human PTH(1-34) that can beemployed in the present method are described in U.S. patent applicationSer. No. 60/069,875, incorporated herein by reference.

Administering Parathyroid Hormone

A parathyroid hormone can typically be administered parenterally,preferably by subcutaneous injection, by methods and in formulationswell known in the art. Stabilized formulations of human PTH(1-34) thatcan advantageously be employed in the present method are described inU.S. patent application Ser. No.60/069,075, incorporated heroin byreference. This patent application also describes numerous otherformulations for storage and administration of parathyroid hormone. Astabilized solution of a parathyroid hormone can include a stabilizingagent, a buffering agent, a preservative, and the like.

The stabilizing agent incorporated into the solution or compositionincludes a polyol which includes a saccharide, preferably amonosaccharide or disaccharide, e.g., glucose, trehalose, raffinose, orsucrose; a sugar alcohol such as, for example, mannitol, sorbitol orinositol, and a polyhydric alcohol such as glycerine or propylene glycolor mixtures thereof. A preferred polyol is mannitol or propylene glycol.The concentration of polyol may range from about 1 to about 20 wt-%,preferably about 3 to 10 wt-% of the total solution.

The buffering agent employed in the solution or composition of thepresent invention may be any acid or salt combination which ispharmaceutically acceptable and capable of maintaining the aqueoussolution at a pH range of 3 to 7, preferably 3-6. Useful bufferingsystems are, for example, acetate, tartrate or citrate sources.Preferred buffer systems are acetate or tartrate sources, most preferredis an acetate source. The concentration of buffer may be in the range ofabout 2 mM to about 500 mM, preferably about 2 mM to 100 mM.

The stabilized solution or composition of the present invention may alsoinclude a parenterally acceptable preservative. Such preservativesinclude, for example, cresols, benzyl alcohol, phenol, benzalkoniumchloride, benzethonium chloride, chlorobutanol, phenylethyl alcohol,methyl paraben, propyl paraben, thimerosal and phenylmercuric nitrateand acetate. A preferred preservative is m-cresol or benzyl alcohol;most preferred is m-cresol. The amount of preservative employed mayrange from about 0.1 to about 2 wt-%, preferably about 0.3 to about 1.0wt-% of the total solution.

Thus, the stabilized teriparatide solution can contain mannitol, acetateand m-cresol with a predicted shelf-life of over 15 months at 5° C.

The parathyroid hormone compositions can, if desired, be provided in apowder form containing not more than 2% water by weight, that resultsfrom the freeze-drying of a sterile, aqueous hormone solution preparedby mixing the selected parathyroid hormone, a buffering agent and astabilizing agent as above described. Especially useful as a bufferingagent when preparing lyophilized powders is a tartrate source.Particularly useful stabilizing agents include glycine, sucrose,trehalose and raffinose.

In addition, parathyroid hormone can be formulated with typical buffersand excipients employed in the art to stabilize and solubilize proteinsfor parenteral administration. Art recognized pharmaceutical carriersand their formulations are described in Martin, “Remington'sPharmaceutical Sciences,” 15th Ed.; Mack Publishing Co., Easton (1975).A parathyroid hormone can also be delivered via the lungs, mouth, nose,by suppository, or by oral formulations.

The parathyroid hormone is formulated for administering a dose effectivefor increasing toughness and/or stiffness of one or more of a subject'sbones and/or for reducing the likelihood and/or severity of bonefracture. Preferably, an effective dose provides an improvement incortical bone structure, mass, and/or strength. Preferably, an effectivedose reduces the incidence of vertebral fracture, reduces the incidenceof multiple vertebral fractures, reduces the severity of vertebralfracture, and/or reduces the incidence of non-vertebral fracture.Preferably, a subject receiving parathyroid hormone also receiveseffective doses of calcium and vitamin D, which can enhance the effectsof the hormone. An effective dose of parathyroid hormone is typicallygreater than about 5 μg/kg/day although, particularly in humans, it canbe as large at about 10 to about 40 μg/kg/day, or larger as is effectiveto achieve increased toughness or stiffness, particularly in corticalbone, or to reduce the incidence of fracture. A subject suffering fromhypoparathyroidism can require additional or higher doses of aparathyroid hormone; such a subject also requires replacement therapywith the hormone. Doses required for replacement therapy inhypoparathyroidism are known in the art. In certain instances, relevanteffects of PTH can be observed at doses less than about 5 μg/kg/day, oreven less than about 1 μg/kg/day.

The hormone can be administered regularly (e.g., once or more each dayor week), intermittently (e.g., irregularly during a day or week), orcyclically (e.g., regularly for a period of days or weeks followed by aperiod without administration). Preferably PTH is administered oncedaily for 1-7 days for a period ranging from 3 months for up to 3 yearsin osteoporotic patients. Preferably, cyclic administration includesadministering a parathyroid hormone for at least 2 remodeling cycles andwithdrawing parathyroid hormone for at least 1 remodeling cycle. Anotherpreferred regime of cyclic administration includes administering theparathyroid hormone for at least about 12 to about 24 months andwithdrawing parathyroid hormone for at least 6 months. Typically, thebenefits of administration of a parathyroid hormone persist after aperiod of administration. The benefits of several months ofadministration can persist for as much as a year or two, or more,without additional administration.

Uses of Formulations of a Parathryoid Hormone

The present invention also encompasses a kit including the presentpharmaceutical compositions and to be used with the methods of thepresent invention. The kit can contain a vial which contains aformulation of the present invention and suitable carriers, either driedor in liquid form. The kit further includes instructions in the form ofa label on the vial and/or in the form of an insert included in a box inwhich the vial is packaged, for the use and administration of thecompounds. The instructions can also be printed on the box in which thevial is packaged. The instructions contain information such assufficient dosage and administration information so as to allow a workerin the field to administer the drug. It is anticipated that a worker inthe field encompasses any doctor, nurse, or technician who mightadminister the drug.

The present invention also relates to a pharmaceutical compositionincluding a formulation of one or more parathyroid hormones, such ashuman PTH(1-84) or human PTH(1-34), and that is suitable for parenteraladministration. According to the invention, a formulation of one or moreparathyroid hormones, such as human PTH(1-84) or human PTH(1-34), can beused for manufacturing a composition or medicament suitable foradministration by parenteral administration. The invention also relatesto methods for manufacturing compositions including a formulation of oneor more parathyroid hormones, such as human PTH(1-84) or humanPTH(1-34), in a form that is suitable for parenteral administration. Forexample, a liquid or solid formulation can be manufactured in severalways, using conventional techniques. A liquid formulation can bemanufactured by dissolving the one or parathyroid hormones, such ashuman PTH(1-84) or human PTH(1-34), in a suitable solvent, such aswater, at an appropriate pH, including buffers or other excipients, forexample to form one of the stabilized solutions described hereinabove.

The examples which follow are illustrative of the invention and are notintended to be limiting.

EXAMPLES Example 1

Increased Bone Strength and Density Upon Administration of rhPTH(1-34)to Rabbits

Experimental Procedures

Female intact New Zealand white rabbits (HRP Inc. Denver, Pa.), one ofthe smallest animals that form osteons by intracortical remodeling,approximately 9 months old and weighing 3.25-3.75 kg, were sorted bymean group body weight into 3 groups of 6 animals each. Two experimentalgroups received biosynthetic PTH(1-34) at doses of 10 or 40μg/ml/kg/day. The control group was given 1.0 ml/kg/day of acidified0.9M saline containing 2% heat-inactivated rabbit sera. PTH(1-34) orvehicle were injected by once daily subcutaneous injections on 5 days aweek for 140 days. Rabbits were fed rabbit lab chow containing 0.5% Caand 0.41%P, and given water ad libitum.

The selection of doses was based on a series of preliminary studiesshowing that (1) after a single injection of PTH(1-34) at 100 pg/kg,serum calcium increased and failed to return to baseline by 24 hours,whereas after a single dose of 50 μg/kg, serum calcium returned tobaseline within 24 hours, (2) repeated injections of 20 μg/kg PTH(1-34)resulted in transient rise in serum calcium with return to baselinevalues in 6-24 hours, and (3) PTH(1-34) at ≦5 μg/kg did not alterhistomorphometry of bone surfaces.

A set of double alizarin labels (Sigma, St. Louis) was given i.m. at 20mg/kg on days 55 and 63, and a set of double calcein labels (Sigma, St.Louis) was given s.c. at 5 mg/kg on days 15 and 7, prior to sacrifice.Rabbits were anesthetized by CO₂ in a random order sequence,approximately 3-6 hours after the last injection, to obtain blood bycardiac puncture and then killed with sodium pentobarbital (100 mg/kg),injected i.p.. The right humerus, both femora, lumbar vertebrae (L3-L5)and the right tibia were removed.

Blood Chemistry

Serum calcium, phosphate, alkaline phosphatase, creatinine and ureanitrogen were measured by computerized multichannel serum analysis.

Histomorphometry

Histomorphometric measurements were done on cortical bone of tibialmidshalt and on cancellous bone of L3. After sacrifice, these bones wereremoved from each animal and fixed in 10% neutral buffered formalin for24 hours. The tissues were dehydrated in a graded series of alcohols(70-100%, 2 changes per grade, each for 4 hours under vacuum). Thespecimens were then placed in xylene, and infiltrated withmethylmethacrylate under vacuum at 20 psi on 2 hours/step and 24 hoursinfiltration schedule in a Shandon Hypercenter automatic processor(Shandon Lipshaw, Pittsburgh, Pa.). The specimens were embedded in 2%DDK-plast with 0.2% initiator (Delaware Diamond Knives, Wilmington,Del.). Cross-sections of tibia were cut at 80 μm using a diamond wiresaw (Delaware Diamond Knives, Inc., Del.) and stained with Goldner'strichrome. Unstained cross sections approximately 80 μm thick wereprocessed for dynamic histomorphometry of fluorochrome labels. Sagittalsections of L3 were cut on Reichert-Jung 2050 microtome (MageeScientific Inc., Dexter, Mich.) at 5 μm and stained with McNeal'stetrachrome, or left unstained for dynamic histomorphometry.

Histomorphometry was done at 150×magnification, using a Nikonfluorescence microscope (Optiphot, Nikon, Tokyo, Japan) and asemi-automatic digitizing system (Bioquant IV, R&M Biometrics,Nashville, Tenn.). Bone formation and resorption in the periosteal,endocortical and intracortical envelopes were measured across the entirecross-sectional area of the mid-diaphyseal sections of the tibia.Measurements on cancellous bone were made within a 6 mm²-area in thecenter of the lumbar vertebra, 0.5 mm from the margin of the surroundingcortical shell. The nomenclature was in accordance with the ASBMRCommittee on histomorphometric nomenclature (Parfitt A M, Drezner M K,Florieux F H, Kanis J A, Malluche H, Meunier P J, Ott S M, Recker R R1987 “Bone histomorphometry: standardization of nomenclature, symbols,and units. Report of the ASBMR Histomorphometry Nomenclature Committee”.J. Bone Miner. Res. 2:595-610.) Dynamic parameters were measured basedon the calcein label.

Bone Mass Measurements The mid-shaft of femora and the fourth lumbarvertebra in 50% ethanol/saline were scanned in cross-section byquantitated computer tomography (QCT or pQCT) employing a 960A pQCT andanalyzed using Dichte software version 5.1 (Norland/Stratec, Ft.Atkinson, Wis.). Whole tissue parameters were measured includingvolumetric bone mineral density (vBMD, mg/cm³), cross-sectional area(X-Area, mm²), and bone mineral content (BMC, mg), using voxeldimensions of 148×148×1200 μm. Volume can be calculated by multiplyingX-Area by the slice thickness of 1.2 mm. The entire femoral neck ofexcised femora in a bath of 50% ethanol, saline were scanned using aperipheral dual energy absorptiometry (pDEXA, Norland/Stratec).Specifically, apparent bone mineral density (aBMD, g/cm²), projectedarea (cm²) and bone mineral content (BMC, g) were measured using scansteps of 0.5×1.0 mm and threshold of 0.04.

Biomechanical Testing

Bone mechanical properties were measured in the right femoral midshaftand the body of L5. Zones were resected, cleaned of connective tissue,wrapped in gauze soaked in isotonic saline and frozen at −20° C. untiltesting. Prior to testing, specimens were thawed for 1-2 hours at roomtemperature. All specimens were tested to failure in a circulating waterbath at 37° C. using an MTS 810 servohydraulic testing machine (MTSCorp., Minneapolis, Minn.). Load-deformation curves were recorded usingthe HP 7090A measurement plotting system (Hewlett Packard, Camas,Wash.). Ultimate force (maximum force that specimens sustain), stiffness(the slope of the linear portion of the load-deformation curve) and workto failure (area under the load-deformation curve before failure) weremeasured using a digitizer system (Jandel Scientific, Corte Madera,Calif.). These parameters are structural properties which depend onintrinsic material properties and geometry. Turner CH, Burr DB 1993“Basic biomechanical measurements of bone: a tutorial.” Bone 14:595-608.The data were normalized to obtain intrinsic material properties such asultimate stress (maximum stress that specimens sustain), elastic modulus(material intrinsic stiffness), and toughness (resistance to fractureper unit volume) which are independent of size and shape. Id.

Femoral bone strength was measured at the midshaft using three-pointbending. The femur was positioned on a fixture with the anterior sidefacing toward the loader. Load was applied on the mid-point between twosupports that were 54 mm apart. The load cell was displaced at the rateof 1 mm/sec until failure occurred. To normalize the data obtained fromthe load-deformation curve, bending ultimate stress was calculated fromultimate force byσ_(f) =FuLr/8I  (1)where σ_(f) is bending fracture stress, Fu is the ultimate force, L isthe length between supports, r is the radius in anterior-posteriordirection, and I is the moment of inertia. Id. The value for the momentof inertia was calculated, assuming that the femoral cross-sections wereelliptical.

Average cortical thickness was calculated from thickness measurementsmade in each of four quadrants of the femoral cross-section with a pairof digital calipers, accurate to 0.01 mm with a precision of ±0.005 mm(Mitutoyo, Japan).

Elastic modulus of the femur (E_(f)) was calculated using the followingequation:E _(f)=(stiffness)*(L³/48I)  (2)

Toughness of the femur (Toughness_(f)) was also calculated using thefollowing equation:Toughness_(f)=3*(work to failure) *r ² /LI  (3)

For the mechanical testing of fifth lumbar vertebra (L5), both endplates of the vertebral body were cut parallel using a Buehler Isometslow speed saw (Buehler LTD, Evanston, Ill.). After resection of theposterior processes, mechanical strength of L5 was measured incompression. The compressive load was applied in stroke control, with across-head speed of 1 mm/sec through a pivoting platen to correct fornonparallel alignment of the faces of the vertebral body. To normalizethe data obtained from the load-deformation curve, and to evaluateintrinsic material properties that are independent of bone geometry,ultimate stress was calculated as the ultimate force divided by thegross cross-sectional area.

Cross-sectional area (CSA) was calculated byCSA=πab/4  (4)where a and b are the width in the anterior-posterior and medial-lateraldirections, respectively).

Elastic modulus of the vertebra (E_(v)) was calculated byE _(v)=(stiffness)/(CSA/h)  (5)where h is the cranio-caudal height of vertebral body.

Toughness of the vertebra (Toughness_(v)) was calculated byToughness_(v)=(work to failure)/)(CSA*h)  (6)Acoustic Microscopy

500-μm thick cross-sections were cut from the mid-diaphysis of the righthumerus using a diamond wire saw. Precise thickness of each specimen wasmeasured using a micrometer (Mitutoyo, Japan) at a resolution of 1 μm.Acoustic velocity measurements were made using a scanning acousticmicroscope (UH3, Olympus, Japan) by the method described previously byHasegawa K, Turner CH, Recker RR, Wu E, Burr DB 1995 “Elastic propertiesof osteoporotic bone measured by scanning acoustic microscopy”. Bone16:85-90. Using this technique, detailed intrinsic mechanical propertiesat a selected focal point can be measured. A 50 MHz transducer (V-390,Panametrics, Waltham, Mass.) was used to generate acoustic waves inpulse-echo mode. The 50 MHz lens produced an acoustic beam,approximately 60 μm in diameter. Specimens were fixed to the bottom of achamber filled with water at constant temperature (22° C.). Delay timebetween acoustic waves reflected from the top of the specimens and thosereflected from the bottom of the specimens was measured using a digitaloscilloscope (TDS 620, Tekronix, Beaverton, Oreg.). Delay times weremeasured at five different locations such that each site was more than300 μm from each other in the anterior cortex of the humerus. Acousticvelocity was calculated as twice the thickness of specimens, divided bythe average delay time. Wet weight (Ww) and submerged weight (Ws) in100% ethyl alcohol were measured using a balance (AJ100, MettlerInstrument Corp., Heightstown, N.J.). Wet density (p) was calculatedusing Archimedes's principle:ρ={Ww/(Ww−Ws)}* ρETOH  (7)where pETOH is the density of alcohol (0.789 g/cm³). Assuming theacoustic wave pathway in bone as homogenous, the elastic coefficient (C)representing the intrinsic stiffness of the specimens is calculated:C=ρ*v²  (8)where ρis wet density and v is acoustic velocity.Statistical Analysis

Bartlett analysis was used to check homogeneity of variance. Whenvariance was homogeneous, one-way ANOVA with Fisher's PLSD tests forpost-hoc comparison was applied. When variance was not homogeneous,Kruskal-Wallis non-parametric analysis of variance was applied, withpost-hoc analysis using Mann-Whitney's U-tests. Statistical significancewas ascribed at p<0.05. Results are presented as mean ±SEM.

Results

Body Weight and Biochemistry

Rabbits treated with vehicle PTH(1-34) at 10 mg/kg/day exhibited minorincrements in body weight over 140 days. Rabbits given PTH(1-34) at 40μg/kg/day exhibited a small decrement of 51 g in body weight,representing a 1.4 ±1.6% loss in body weight during the experiment(Table 1). Serum measures were within the normal physiological responsefor rabbits, although small increases in serum calcium and urea nitrogenwere observed. Serum alkaline phosphatase increased by 2-fold at thehigher PTH(1-34) dose (Table 2). TABLE 1 Effects of PTH(1-34) on BodyWeight PTH(1-34) PTH(1-34) control 10 μg/kg/day 40 μg/kg/day Initialbody weight (kg) 3.43 ± 0.08 3.42 ± 0.08 3.42 ± 0.08 Final body weight(kg) 3.70 ± 0.05 3.51 ± 0.05 3.37 ± 0.10 Body weight gain (kg) 0.26 ±0.09 0.09 ± 0.05 −0.05 ± 0.05*Data are expressed as mean ± SEM for 6 rabbits per group.*P < 0.05 compared with control.

TABLE 2 Effects of PTH(1-34) on Serum Chemistry PTH(1-34) PTH(1-34)control 10 μg/kg/day 40 μg/kg/day Calcium (mg/dl) 12.1 ± 0.3 12.6 ± 0.213.5 ± 0.3* Phosphate (mg/dl)  4.7 ± 0.2  4.7 ± 0.2 5.5 ± 0.3 Alkalinephosphatase (iu/l) 24.7 ± 4.1 41.0 ± 8.1 49.8 ± 7.1* Creatinine (mg/dl) 1.9 ± 0.1  1.6 ± 0.1 1.8 ± 0.1 Urea nitrogen (mg/dl) 18.3 ± 0.3 18.1 ±0.8 23.9 ± 1.9 Data are expressed as mean ± SEM for 6 rabbits per group.*P < 0.05 compared with control.Histomorphometry

Bone formation on periosteal (Ps.MS/BS) and endocortical (Ec.MS/BS)surfaces of the tibial midshaft increased in PTH(1-34) treated groups(Table 3). Ps.MS/BS in the higher dose group was significantly greaterthan in the other 2 groups (p<0.001) and Ec.MS/BS in the higher dosegroup was significantly greater than in the control group (p<0.05).Consistent with the increase in serum alkaline phosphatase, boneformation rates on each surface (Ps.BFR/BS and Ec.BFR/BS) weresignificantly greater in the higher dose group than the other 2 groups(p<0.05). Mineral apposition rate (MAR) did not change on eitherperiosteal or endocortical envelopes.

Intracortically, the number of resorption sites (Rs.N/Ct.Ar) in rabbitsgiven PTH(1-34) at 40 μg/kg/day was significantly greater by 7-fold,than in the other 2 groups (p<0.05) (Table 4). The number of labeledosteons (L.On.N/Ct.Ar) in rabbits given PTH(1-34) at 40 μg/kg/day alsosignificantly increased compared to the other 2 groups (p<0.01 vs thecontrol group, p<0.05 vs 10 μg/kg/day group). MAR was significantlygreater in both treatment groups than in the control group (p<0.01), butthere was no significant difference between the PTH-treated groups. Boneformation rate (BFR/BV) and activation frequency (Ac.F) increased(p<0.05 and p<0.01, respectively) at both doses.

Although bone area (B.Ar) increased at both doses, a significantdifference was only found between the higher dose group and the controlgroup (p<0.01). Marrow area (Ma.Ar) decreased after treatment, but wasnot significantly different among the three groups. However, corticalarea (Ct.Ar) in the higher dose group was significantly greater than theother 2 groups (p<0.0001 vs the control group, p<0.05 vs the lower dosegroup). Ct.Ar in the lower dose group was also significantly higher thanthe control (p<0.05). Similar results were found in %Ct.Ar.

Cortical porosity (Ct.Po) in rabbits given PTH(1-34) at 10 μg/kg/day wasdouble that in the control group (p<0.05), while Ct.Po in rabbits givenPTH(1 -34) at 40 μg/kg/day was 6× higher than the control group (p<0.01)However, porosities lay within the endocortical compartment and, withinthat location, are unlikely to contribute to biomechanical strength asPTH also increased cortical bone area, consistent with an increase incross-sectional moment of inertia. TABLE 3 Effects of PTH(1-34) onperiosteal and endocortical bone remodeling of tibial midshaft.PTH(1-34) PTH(1-34) Parameters Abbreviation control 10 μg/kg/day 40μg/kg/day Endocortical osteoid surface Ec.OS/BS (%) 8.8 ± 6.0 13.7 ±10.5 20.2 ± 5.8  Endocortical osteoid thickness Ec.O.Th (μm) 7.4 ± 2.43.7 ± 2.3 8.1 ± 0.9 Periosteal mineral apposition rate Ps.MAR (μm/day)0.33 ± 0.17 0.38 ± 0.08 0.66 ± 0.14 Endocortical mineral apposition rateEc.MAR (μm/day) 1.33 ± 0.22 0.79 ± 0.16 1.32 ± 0.15 Periostealmineralizing surface Ps.MS/BS (%) 3.8 ± 1.9 8.2 ± 2.1 22.3 ± 2.7*^(‡)Endocortical mineralizing surface Ec.MS/BS (%) 26.4 ± 6.6  32.6 ± 8.2  57.7 ± 10.4* Periosteal bone formation rate Ps.BFR/BS 0.02 ± 0.02 0.03± 0.01  0.16 ± 0.05*‡ (μm³/μm²/yr) Endocortical bone formation rateEc.BFR/BS 0.40 ± 0.10 0.31 ± 0.10  0.72 ± 0.12*^(‡) (μm³/μm²/yr)Data are expressed as mean ± SEM for 6 rabbits per group.*P < 0.05 compared with control.^(†)P < 0.05 compared with PTH(1-34) 10 μg/kg/day.

TABLE 4 Effects of PTH(1-34) on intracortical bone remodeling of tibialmidshaft. PTH(1-34) PTH(1-34) Parameters Abbreviation control 10μg/kg/day 40 μg/kg/day Resorption cavity number Rs.N/Ct.Ar (#/mm²) 0.014± 0.013 0.013 ± 0.004  0.097 ± 0.036*^(‡) Labeled osteon numberL.On.N/Ct.Ar (#/mm²) 0.011 ± 0.006 0.027 ± 0.006  0.215 ± 0.094*‡Osteoid thickness O.Th (μm) 4.92 ± 0.59 5.42 ± 0.30 5.16 ± 0.27 Mineralapposition rate MAR (μm/day) 1.19 ± 0.20  1.56 ± 0.13*  1.60 ± 0.12*Bone formation rate BFR/BV (%/yr) 0.5 ± 0.3  8.5 ± 2.9* 21.4 ± 3.8*Activation frequency Ac.F (#/mm²/yr) 1.8 ± 1.0 15.1 ± 5.0*  43.8 ±10.5*^(‡) Bone area B.Ar (mm²) 29.1 ± 1.3  33.3 ± 1.9  37.8 ± 2.7*Marrow area Ma.Ar (mm²) 12.7 ± 0.7  11.9 ± 1.0  10.7 ± 1.0  Corticalarea Ct.Ar (mm²) 16.4 ± 0.9  21.3 ± 1.2* 27.1 ± 2.0*^(‡) % Cortical area% Ct.Ar (%) 56.4 ± 1.5  64.2 ± 1.6* 71.6 ± 1.5*Data are expressed as mean ± SEM for 6 rabbits per group.*P < 0.05 compared with control.^(‡)P < 0.05 compared with PTH(1-34) 10 μg/kg/day.

TABLE 5 Effects of PTH(1-34) on cancellous bone remodeling of thirdlumbar vertebra. PTH(1-34) hPTH(1-34) Parameters Abbreviation control 10μg/kg/day 40 μg/kg/day Bone volume BV/TV (%) 27.5 ± 1.4  30.5 ± 3.4 27.9 ± 3.2  Trabecular thickness Tb.Th (μm) 124.8 ± 7.3  147.4 ± 12.7 126.4 ± 13.7  Eroded surface ES/BS (%) 0.5 ± 0.3 1.4 ± 0.3  2.6 ± 0.7*Osteoclast surface Oc.S/BS (%) 0.4 ± 0.2 0.9 ± 0.3 1.3 ± 0.3 Osteoidsurface OS/BS (%) 5.2 ± 1.3 7.2 ± 1.2 27.7 ± 3.8*^(‡) Osteoblast surfaceOb.S/BS (%) 1.4 ± 0.6 1.3 ± 0.6 15.3 ± 5.6*‡ Osteoid thickness O.Th (μm)5.2 ± 0.5 5.3 ± 0.5 4.4 ± 0.2 Osteoid volume OV/TV (%) 0.10 ± 0.02 0.13± 0.03  0.46 ± 0.07*^(‡) Mineral apposition rate MAR (μm/day) 1.3 ± 0.21.5 ± 0.1 1.7 ± 0.1 Mineralizing surface MS/BS (%) 4.4 ± 1.4 7.4 ± 1.924.2 ± 1.5*‡ Bone formation rate BFR/BS (μm³/μm²/yr) 19.7 ± 5.3  38.5 ±8.9  153.0 ± 15.6*^(‡)Data are expressed as mean ± SEM for 6 rabbits per group.*P < 0.05 compared with control.^(‡)P < 0.05 compared with PTH(1-34) 10 μg/kg/day.

In cancellous bone, most of the formation parameters (OS/BS, Ob.S/BS,OV/TV, and MS/BS) increased with PTH(1-34) treatment (Table 5). Those inrabbits given PTH(1-34) at 40 pg/kg/day were significantly greater thanin the other 2 groups (p<0.01 vs both the control group and 10 μg/kg/daygroup in all parameters). Bone formation rate (BFR/BS) alsosignificantly increased in rabbits given PTH(1-34) at 40 μg/kg/daycompared to the other 2 groups (p<0.0001 vs both the control and 10μg/kg/day,groups). Although, resorption (ES/BS and Oc.S/BS) increased inboth PTH(1-34) treated groups, only eroded surface (ES/BS) in the higherdose group was significantly greater than the control group (p<0.001).There were no differences in osteoid thickness (O.Th) among the threegroups. Despite the evidence for accelerated bone turnover, fractionalbone volume (BV/TV) did not change after PTH(1-34) treatment. Tunnelingresorption and peritrabecular fibrosis were not observed in any of thegroups.

Bone Mass Measurements

vBMD and BMC in the midshaft of the femur assessed by pQCT in 40μg/kg/day group were significantly higher than in the other 2 groups(p<0.001 in vBMD and p<0.0001 in BMC vs the control group, p<0.05 invBMD and p<0.01 in BMC vs the lower dose group) (FIG. 1A). vBMD and BMCin 10 μg/kg/day group were also significantly higher than in the controlgroup (p<0.05 in both vBMD and BMC). Although bone area of the midshaftof the femur also increased dose-dependently, it significantly increasedonly in 40 μg/kg/day group (p<0.05).

aBMD and BMC in the proximal femur, measured by dual X-rayabsorbtiometry (DXA or pDXA), increased dose-dependently. Significantdifferences were present in both aBMD and BMC between the control groupand 10 μg/kg/day group (p<0.05) as well as between the control group and40 μg/kg/day group (p<0.001) (FIG. 1B). No significant differences werefound in bone area among the three groups.

Overall, FIG. 1 shows that BMD (bone mineral density) and BMC (bonemineral content) in the femoral midshaft (cortical bone) (A) and in theproximal femur (cancellous bone +cortical bone) (B) were significantlygreater in PTH-treated animals than controls at both doses. Corticalbone area at the femoral midshaft in rabbits treated at the higher dosewas significantly greater than controls. No significant differences werefound between groups in bone area of the proximal femur. Data areexpressed as mean ±SEM. *P<0.05 compared with the control. ‡P<0.05compared with PTH 10 μg/kg/day.

There were no significant differences in vBMD, BMC or bone area of thelumbar vertebra (L4) assessed by pQCT, among the three groups.

Biomechanical Testing

Structural properties of the midshaft of the femur, such as ultimateforce, stiffness and work to failure increased dose-dependently (FIG.2). FIG. 2 shows the effects of PTH on mechanical strength and crosssectional moment of inertia (CSMI) in the cortical bone of the femoralmidshaft. Structural mechanical properties (open bars) and CSMIincreased significantly in the higher dose group, while stiffness alsoincreased significantly in the lower dose group. Of the intrinsicmaterial properties (dark bars), only elastic modulus increasedsignificantly in the lower dose group when compared to controls. Elasticmodulus in the higher dose group was significantly decreased whencompared to the lower dose group. In FIG. 2: data are expressed as mean±SEM; * indicates P<0.05 compared with the control; and ‡indicatesP<0.05 compared with 10 μg/kg/day.

In this study and the results shown in FIG. 2, all parameters weresignificantly higher in rabbits given PTH(1-34) at 40 μg/kg/day than inthe control group (p<0.01 for ultimate force and work to failure, p<0.05for stiffness). Stiffness in the lower dose group was also significantlyhigher than in the control group (p<0.05). Of the intrinsic materialproperties, elastic modulus was significantly less in rabbits given 40μg/kg/day than those given 10 μg/kg/day (p<0.01).

In the lumbar vertebral body, no significant differences were found inmechanical properties among the three groups.

Acoustic Microscopy

There were no significant differences in acoustic velocity or elasticcoefficient among the three groups.

Discussion

The skeletal response of cortical bone to biosynthetic hPTH(1-34)involved both a direct regulation of material properties and acompensatory regulation of biomechanical properties in the long bones ofintact, mature female rabbits. PTH(1-34) increased bone turnover andcortical porosity and, at the 40 μg/kg dose, reduced the materialelastic modulus of cortical bone. However, the decreased elastic moduluswas more than compensated by increased bone apposition on periosteal andendocortical surfaces, resulting in a significant improvement in thestructural strength, stiffness and work to failure of cortical bone inrabbits.

In this study using intact rabbits, cancellous bone volume of the lumbarvertebra did not change after PTH(1-34) treatment despite the evidencefor increased bone turnover. Previous use as an osteopenic model,presence of intracortical remodeling, and short remodelingperiod—together with the rabbit's rapid growth and early skeletalmaturation (by 6-9 months), formed the basis for selection of the rabbitas a model in which to test the effects of intermittently administeredPTH(1-34).

Rabbits can exhibit a wide variation in serum calcium levels (10-16mg/dl), but these levels are not directly influenced by the amount ofdietary calcium, another advantage of the model. Although transientsignificant increases of approximately 1 mg/ml were recorded in rabbitstreated with PTH(1-34) at 40 μg/kg, the actual values were always withinthe known physiologic range.

In the current study, biosynthetic hPTH(1-34) for 140 days increasedbone formation rate intracortically as well as on periosteal andendocortical surfaces. Intracortical Ac.F increased in the lower dosegroup by 8× and in the higher dose group by 20×. This led to a 2-foldincrease in cortical porosity in the tibia in the lower dose group and a6-fold increase in the higher dose group. The data from acousticmicroscopy shows that elastic properties of the bone material itself inthe humerus was not affected, indicating that intrinsic cortical bonequality is normal. Therefore, the increased porosity must account forthe slight reduction in elastic modulus, a material propertiesmeasurement that includes the spaces in the cortex.

Increased cortical porosity was more than compensated, however, bysignificantly increased MS/BS and BFR/BS on both periosteal andendocortical surfaces in the midshaft of the tibia in the higher dosegroup, resulting in significantly increased bone area. This wouldincrease the cross-sectional moment of inertia, which is proportional tothe bone's bending rigidity, as it did in the femoral midshaft (FIG. 2).The consequence of these changes in shape and material properties was toimprove the mechanical strength and stiffness of the femoral diaphysiswhen compared to controls, thus offsetting the potentially deleteriousmechanical effects of increased cortical porosity.

Conclusion

In conclusion, the increases in bone turnover and cortical porosityafter PTH(1-34) treatment were accompanied by concurrent increases inbone at periosteal and endocortical surfaces. The combination of thesephenomena resulted in an enhancement of the toughness ultimate stress,stiffness, and work to failure of the femur.

Example 2

Increased Bone Strength and Density Upon Administration of rhPTH(1-34)to Monkeys

Experimental Procedures

General

The live phase of the study used feral, adult (closed growth plates)cynomolgus primates (Macaca fascicularis), weighing 2.77±0.03 kg (mean±standard error of the mean [SEM]). Monkeys were held in quarantine for3 months, then started on a diet containing 0.3% calcium, 0.3%phosphate, and 250 IU vitamin D3/100 g diet, and given fluoridated water(1 ppm fluoride) ad libitum. The calcium content corresponded to 1734 mgcalcium/2000 calories. After 1 month on the diet, animals were sortedinto groups of 21 or 22, sham operated or ovariectomized. Once dailysubcutaneous injections of vehicle (sham and ovariectomized controls) orrhPTH(1-34), at 1 μg/kg (PTH1) or 5 μg/kg (PTH5), were started 24 hoursafter ovariectomy. Animals were treated for either 18 months (PTH1 andPTH5), or for 12 months followed by withdrawal of treatment (PTH1-W andPTH5-W).

The study groups were divided as shown in Table 6. TABLE 6 Study Groupsfor Primate Study Monkeys Monkeys in at Final Outset Analyses GroupAbbreviation (n = 128) (n = 121) Sham ovariectomized, 18 months vehicleSham 21 21 Ovariectomized, 18 months vehicle OVX 22 20 Ovariectomized,18 months 1 μg rhPTH(1-34)/kg/day PTH1 21 19 Ovariectomized, 12 months 1μg rhPTH(1-34/kg/day, 6 PTH1-W 21 20 months vehicle Ovariectomized, 18months 5 μg rhPTH(1-34)/kg/day PTH5 22 21 Ovariectomized, 12 months 5 μgrhPTH(1-34)/kg/day, 6 PTH5-W 21 20 months vehicle

Serum and urinary samples were taken 24 hours after vehicle orrhPTH(1-34) injection at 3-month intervals. A sparse sampling design of5 monkeys in each rhPTH(1-34)-treated group was used forpharmacokinetics, with sampling (spanning 0 to 240 minutes each time) atbaseline, 7, 11, and 17 months. At 0 time and at 6-month intervals,total skeleton and spine (L-2 to L-4) bone mass were assessed bydual-energy x-ray absorptiometry (DXA); peripheral quantitative computedx-ray tomography (pQCT) was used to assess bone mass in the midshaft anddistal radii, and the proximal tibia. Iliac biopsies were taken at 6 and15 months for histomorphometry. All animals were euthanized after 18months.

Biomechanical tests were done on lumbar vertebrae L-3 to L-4, the femurneck, humerus midshaft, and on a cortical bone specimen machined fromthe femur diaphysis (measures defined in Table 7). Conventional staticand dynamic histomorphometry were done (measures described in Table 11)on the humerus midshaft, lumbar vertebra L-2, femoral neck, femurmidshaft, the radius midshaft and distal radius. Initial statisticalanalyses compared all groups to vehicle-treated ovariectomized controls.The data is suitable for additional exploratory analyses to examine dosedependency, effects of withdrawal, interactions between outcomes, andchanges in time by methods known to those of skill in the art. Allassays were conducted and determined by methods known in the art.

For certain experimental subjects, cortical bone of the humerus wasexamined by histomorphometry and by polarized Fourier transform infraredmicroscopy. The Fourier transform infrared microscopy was conducted byan adaptation of known methods for such microscopy.

3D Finite Element Modeling Studies

These studies determined 3D finite element modeling data on vertebrafrom monkeys of the study dosed with PTH for 18 months. Excised L-5vertebra in 50% ethanol/saline from the ovariectomized (n=7) and PTH(n=7) groups were serially scanned in 500 μm steps by quantitativecomputed tomography (QCT, Norland, Ft. Atkinson. Wis.), using 70×70 μmpixels. Each of the 500 μm cross-sections was analyzed for volumetricbone mineral density (BMD, mg/cc), bone mineral content (BMC, mg),cross-sectional area (X-Area), cancellous bone volume (BV/TV),trabecular thickness (Tb.Th), and connectivity (node density, strutanalysis). Pixels in each serial scan were averaged to create490×490×500 μm voxels. The serial scans were then stacked and atriangular surface mesh generated for each bone using the “marchingcubes” algorithm (see e.g. Lorensen and Cline 1987 “Marching cubes, ahigh resolution 3D surface construction algorithm.” Computer Graphics21, 163-169). A smoothed version of each surface mesh was then used togenerate a tetrahedral mesh for 3D finite-element modeling.

Young's modulus for each tetrahedral element was derived from theoriginal voxel densities and material properties from a beam of corticalbone milled from the femoral diaphysis of the monkeys. Each tetrahedralmesh was rotated so that the bottom surface of each vertebra was alignedwith a plane. Linear elastic stress analysis was then performed for eachL-5 model in which a distributed load of 100 N was applied to the topsurface of the centrum, perpendicular to the bottom plane while thebottom surface was fixed in the direction of loading. The resultingaxial strain contours were evaluated, as were the BMD distributions, andcompared between PTH and ovariectomized. At this resolution, the densityof each voxel is dependent on the extent to which each voxel is filledwith bone as opposed to soft tissue.

Results

Reports of differences in the text are statistically significant,p<0.05. All animals gained 4% to 9% of initial body weight during thestudy independent of treatment.

Serum and Urine Measures

Serum estradiol levels at 3 and 18 months were below 5 pg/mL in allovaricctomized monkeys. When measures of calcium homeostatis werecompared to sham controls, ovariectomized controls had lower serumcalcium and phosphate and 1,25-dihydroxyvitamin D levels, but did notdiffer in endogenous PTH, urinary cyclic adenosine monophosphate (cAMP),urinary calcium, urinary creatinine, or serum urea nitrogen measured 24hours after last injection. Animals treated with rhPTH(1-34) had lowerserum phosphate, lower endogenous PTH, and higher 1,25-dihydroxyvitaminD and urinary cAMP compared to ovariectomized. Serum bone formationmarker assays showed that ovariectomized monkeys had low serum totalalkaline phosphatase (ALP) and osteocalcin compared to shams, andrhPTH(1-34) restored levels back to sham values. Urinary C-telopeptide(CrossLaps) excretion, used as a biochemical marker of bone resorption,was not altered by rhPTH(1-34) compared to ovariectomized controls.

Bone Mass

Overall skeletal bone mass, expressed as total body BMC, was increasedsignificantly by PTH(1-34) (FIG. 3). Spine bone mineral density (BMD)remained stable in ovariectomized controls for 18 months, while shamcontrols gained approximately 5% above baseline (FIGS. 4A-4C and 5A).rhPTH(1-34) increased spine BMD by 7% to 14% and whole body bone mineralcontent (BMC, FIG. 3) by up to 6% compared to baseline (FIGS. 4A-4C and5A). Spine bone mineral content also increased (FIG. 5A). inrhPTH(1-34)-treated primates, the magnitude of these increases wassignificantly higher than that of ovariectomized controls, and matched(PTH1) or exceeded (PTH5) that of shams. rhPTH(1-34) did not alter BMDof the midshaft or distal radius. The cross-sectional area of themidshaft increased by 7% in the PTH5 group. In the proximal tibia, therewas no increase in cross-sectional area but rhPTH(1-34) increased BMCand BMD compared to ovariectomized controls. Six months after treatmentwas withdrawn, BMD and BMC in the spine and femur neck remained higherthan ovariectomized controls, with no change in the cortical midshaft ofthe humerus.

Bone Strength

rhPTH(1-34) increased strength (F_(y)) in the vertebrae by up to 43%(Tables 7 and 8, FIG. 5B). rhPTH(1-34) improved strength in the femurneck (f_(u)) by up to 12% (Tables 7 and 9, FIG. 6A). rhPTH(1-34) did notalter measures in the cortical diaphysis of the humerus midshaft (Tables7 and 10), or the material properties of beam specimens machined fromthe femoral diaphysis (Tables 7 and 9, FIG. 6B) when compared toovariectomized controls. In animals treated with rhPTH(1-34) for 12months and then withdrawn from treatment for 6 months, bone strengthmeasures remained significantly higher than ovariectomized controls(Tables 7-10, FIGS. 5B and 6A). TABLE 7 Variables Reported for the Thirdand Fourth Lumbar Vertebrae (L-3 and L-4), Humerus Midshaft, ProximalFemoral Neck, and Femoral Beam Specimens Variable Units DescriptionLumbar Vertebrae, L-3 and L-4 A mm² Cross-sectional area F N Yield forceis the force at a 0.2% offset S N/mm Slope of the linear portion of theforce-displacement curve (stiffness) σγ Mpa Yield stress E Mpa Young'smodulus Humerus Midshaft t mm Average cortical thickness F_(u) NUltimate force is the maximum force the specimen can withstand S N/mmSlope of the linear portion of the force-displacement curve (stiffness)here's stiffness mJ/U N-mm Area under the load-displacement curve (U =work to failure) Proximal Femoral Neck F_(u) N Ultimate force is themaximum force the specimen can withstand Femur Diaphysical BeamSpecimens σ_(u) Mpa Ultimate stress E Gpa Young's modulus u J/m³Toughness ε_(u) Ultimate strain

TABLE 8 Biomechanical Measures of Strength in the Spine (LumbarVertebrae, L-3 and L-4 Combined) of Ovariectomized Primates at 18 MonthsVariable (units)^(a) Sham OVX Control PTH1 PTH1-W PTH5 PTH5-W A (mm²) 90.5 ± 2.1^(b) 86.7 ± 2.3 88.3 ± 2.0  90.9 ± 2.3  87.3 ± 2.7  82.8 ±2.1  F_(v) (N) 1738 ± 52  1499 ± 94^(s) 1915 ± 105^(o) 1899 ± 73^(o)  2113 ± 77^(s,o) 1792 ± 59^(o)  S (N/mm) 7312 ± 319  5805 ± 476^(s) 7701± 474^(o) 7401 ± 452^(o) 8012 ± 367^(o) 7074 ± 314^(o) σ_(v) (Mpsa) 19.4± 0.6 17.3 ± 1.0 21.9 ± 1.3^(o) 21.1 ± 0.8^(o)   24.6 ± 1.1^(s,o) 21.9 ±0.9^(o) E (Mpa) 650 ± 32 546 ± 49 717 ± 48^(o) 659 ± 42  759 ± 36^(o)698 ± 41^(o)Abbreviations:OVX = ovariectomized;PTH1 = rhPTH(1-34) 1 μg/kg for 18 months;PTH1-W = withdrawal for 6 months after treatment with rhPTH(1-34) 1μg/kg for 12 months;PTH5 = rhPTH(1-34) 5 μg/kg for 18 months;PTH5-W = withdrawal for 6 months after treatment with rhPTH(1-34) 5μg/kg for 12 months.^(a)See Table 4.1 for description of variables^(b)Data expressed as mean ± standard error of the mean (SEM) per group.^(o)Statistically significant compared to OVX controls (p < 0.05).^(s)Statistically significant compared to sham controls (p < 0.05).

TABLE 9 Biomechanical Measures of Material Properties of Equivalent SizeBeam Specimens from Femur Diaphysis, and Biomechanical Measure ofStrength of Femur Neck of Ovariectomized Primates at 18 Months Variable(units)^(a) Sham OVX Control PTH1 PTH1-W PTH5 PTH5-W σ_(u) (Mpa) 222 ±5^(b ) 216 ± 5  222 ± 4  214 ± 6  206 ± 6  208 ± 6  E (Gpa) 17.2 ± 0.6 16.4 ± 0.4  17.1 ± 0.4  16.6 ± 0.6  15.4 ± 0.6^(s)   15.3 ± 0.6^(s) u5.9 ± 0.3 5.8 ± 0.4 6.1 ± 0.4 5.5 ± 0.4 5.4 ± 0.4 6.1 ± 0.4 (mJ/m³)ε_(u) 0.035 ± 0.001 0.035 ± 0.002 0.036 ± 0.002 0.034 ± 0.002 0.034 ±0.002 0.038 ± 0.002 Proximal femur neck F_(u) 1288 ± 41  1105 ± 53^(s )1235 ± 45^(o )  1258 ± 52^(o ) 1362 ± 30^(o)  1213 ± 42 Abbreviations:OVX = ovariectomized;PTH1 = rhPTH(1-34) 1 μg/kg for 18 months;PTH1-W = withdrawal after treatment with rhPTH(1-34) 1 μg/kg for 12months;PTH5 = rhPTH(1-34) 5 μg/kg for 18 months;PTH5-W = withdrawal after treatment with rhPTH(1-34) 5 μg/kg for 12months.^(a)See Table 4.1 for description of variables^(b)Data expressed as mean ± standard error of the mean (SEM) per group.^(o)Statistically significant compared to OVX controls (p < 0.05).^(s)Statistically significant compared to sham controls (p < 0.05).

TABLE 10 Biomechanical Measures of Cortical Bone of the Midshaft of theHumerus of Ovariectomized Primates at 18 Months Variable (units)^(a)Sham OVX Control PTH1 PTH1-W PTH5 PTH5-W t(mm)   1.74 ± 0.04^(b)   1.63± 0.03^(s)  1.68 ± 0.03  1.66 ± 0.04   1.80 ± 0.04^(o)  1.72 ± 0.05F_(u)(N) 725 ± 26 636 ± 26 654 ± 23 689 ± 23  680 ± 15^(s) 707 ± 24S(N/mm) 601 ± 23 520 ± 26 544 ± 23 573 ± 20 548 ± 18 573 ± 24 U(mJ) 1797± 85  1542 ± 92  1641 ± 137 1751 ± 84  1804 ± 99  1775 ± 113Abbreviations:OVX = ovariectomized;PTH1 = rhPTH(1-34) 1 μg/kg for 18 months;PTH1-W = withdrawal after treatment with rhPTH(1-34) 1 μg/kg for 12months;PTH5 = rhPTH(1-34) 5 μg/kg for 18 months;PTH5-W = withdrawal after treatment with rnPTH(1-34) 5 μg/kg for 12months.^(a)See Table 4.1 for description of variables^(b)Data expressed as mean ± standard error of the mean (SEM).^(o)Statistically significant compared to OVX controls (p < 0.05).^(s)Statistically significant compared to sham controls (p < 0.05).Bone Histomorphometry

Although turnover rates were greater in ovariectomized than shamcontrols, there was no significant loss of bone volume in the iliaccrest. As the tetracycline label given at 6 months was not detectable inmany animals, only static parameters were measured for this time point.Static and dynamic histomorphometry data at 15 months showed thattreatment with rhPTH(1-34) increased cancellous bone area compared toovariectomized, and increased bone formation without increasingresorption measures above those measured in ovariectomized controls.Bone formation rate was increased progressively by higher doses ofrhPTH(1-34). Although cancellous bone remained increased compared toovariectomized controls after withdrawal of rhPTH(1-34) following 12months of treatment, bone formation and resorption reverted to that seenin ovariectomized controls, and bone turnover remained higher than insham controls. rhPTH(1-34) did not affect mineralization, activationfrequency, or remodeling periods. There were no differences inindividual bone multicellular unit (BMU)-based bone balance betweenresorption and formation. In summary, rhPTH(1-34) increased cancellousbone by selective stimulation of bone formation.

In the cortical bone of the humerus, where rhPTH(1-34) did notsignificantly modify BMD or bone strength measures, rhPTH(1-34)stimulated changes in the periosteal, endosteal, and intracorticalcompartments (Tables 11 and 12). Although there were no differences intotal area or medullary area between groups, rhPTH(1-34) increasedcortical area, and the PTH5 and PTH5-W groups had significantly morecortical bone, suggestive of increased cross-sectional moment ofinertia, a measure of strength. The increase in area could be attributedto increased formation on both periosteal and endosteal surfaces (FIG.7).

Sham controls and PTH5-W groups had reduced periosteal mineralizingsurfaces compared to ovariectomized controls and the otherrhPTH(1-34)-treated groups. Endocortical mineralizing surfaces weresignificantly greater in ovariectomized controls compared to shams andrhPTH(1-34) did not increase above ovariectomized control values. Inintracortical remodeling, there were more resorption spaces inovariectomized animals, and activation frequency was greater inovariectomized, PTH1, and PTH5 groups than in sham controls or either ofthe withdrawal groups. There were significantly more labeled osteons perunit area in ovariectomized compared to sham controls, and rhPTH(1-34)did not increase these significantly above ovariectomized controlvalues.

Intracortical porosity was greater in ovariectomized compared to shams,but not different between ovariectomized controls and PTH1. PTH5 andPTH5-W increased porosity above that seen in ovariectomized controls.Data from the rabbit studies suggested the hypothesis that the increasein porosity, accompanied by increased cortical bone, may be a structuralresponse to maintain the biomechanical properties of rhPTH(1-34)-treatedbone. There were no differences between ovariectomized and the othergroups in formation period, osteoid width, wall width, or osteoidmaturation at 18 months.

In summary, there were no differences in turnover rates betweenovariectomized controls and either dose of rhPTH(1-34). Sham controlshad a lower turnover rate than either ovariectomized controls or rhPTH(1-34)-treated animals. When rhPTH(1-34) was withdrawn for 6 months,turnover rates decreased significantly, but BMD and biomechanicalstrength measures remained higher than ovariectomized controls. Normalvalues for osteoid width and maturation time intracortically for allgroups indicates treatment did not cause any defect in the normal timingof the mineralizing process. Normal values for wall width indicate thattreatment did not alter the normal balance between resorption andformation at the level of the individual BMU. TABLE 11 HistomorphometricVariables for Cortical Bone Measurements of the Humerus Variable^(a)Units Description Ac F cycles/year Activation frequency BFR/BS Ec μm/dayBone formation rate, endocortical surface referent BFR/BS.Ps μm/day Boneformation rate, periosteal surface referent BFR/BV %/year Bone formationrate, bone volume referent FP days Formation period L On N/Ct A #/mm²Number of fluorochrome labeled osteons per unit cortical area MAR μm/dayMineral apposition rate, intracortical MAR Ec μm/day Mineral appositionrate, endocortical surface MAR Ps μm/day Mineral apposition rate,periosteal surface MS/BS.Ec % Mineralizing endocortical surfacenormalized to total endocortical surface MS/BS.Ps % Mineralizingperiosteal surface normalized to total periosteal surface O W₁ μmOsteoid width Rs N/Ct A #/mm² Number of resorption spaces per unitcortical area W W₁ μm Osteonal wall width omt days Osteoid maturationtime Po % Porosity, the percentage of bone area occupied by spaces B Armm² Bone area, the total area within the periosteal surface Ct Ar mm²Cortical area, the area of bone within the periosteal surface (includesporosities) Mc Ar mm² Medullary cavity area^(a)Nomenclature is that recommended in Journal of Bone and MineralResearch, 1987.

TABLE 12 Cortical Histomorphometry of the Humerus Midshaft ofOvariectomized Primates at 18 Months (n = 121) Variable^(b) Sham OVXPTH1 PTH1-W PTH5 PTH5-W Ac.F   1.85 ± 1.87^(a,c) 6.06 ± 3.31 7.69 ± 4.96 3.05 ± 2.15^(a) 8.70 ± 3.97  2.05 ± 1.46^(a) BFR/BS.Ec 7.08 ± 3.80 20.93 ± 19.23 18.14 ± 13.95 14.89 ± 10.32 34.04 ± 19.01  12.73 ±16.33^(a) BFR/BS.Ps 3.79 ± 3.07  9.12 ± 7.58  8.53 ± 10.54  3.60 ±3.84^(a) 8.99 ± 5.81 5.79 ± 4.05 BFR/BV 2.13 ± 2.06^(a) 9.16 ± 5.37 9.23± 5.93  4.39 ± 3.48^(a) 12.93 ± 5.94^(a)  2.21 ± 1.73^(a) FP 82.73 ±41.06  65.94 ± 19.79 63.44 ± 10.02 64.94 ± 11.99 81.97 ± 95.63 88.63 ±52.90 L.On.N/Ct.A 0.28 ± 0.27^(a) 1.03 ± 0.52 1.26 ± 0.71  0.50 ±0.36^(a)  1.45 ± 0.47^(a)  0.38 ± 0.26^(a) MAR 0.91 ± 0.33^(a) 1.07 ±0.19  0.98 ± 0.12^(a) 1.06 ± 0.27 1.03 ± 0.23  0.85 ± 0.28^(a) MAR.Ec0.48 ± 0.19^(a) 0.75 ± 0.25 0.66 ± 0.15 0.66 ± 0.16 0.75 ± 0.14 0.63 ±0.17 MAR.Ps 0.62 ± 0.24  0.69 ± 0.23 0.89 ± 0.95  0.54 ± 0.15^(a) 0.66 ±0.17 0.82 ± 0.15 MS/BS.Ec 3.09 ± 6.49^(a) 20.99 ± 18.04 25.19 ± 17.14 11.74 ± 14.41^(a)  40.47 ± 24.68^(a)   8.93 ± 15.39^(a) MS/BS.Ps 1.81 ±3.59^(a) 10.03 ± 10.49 8.59 ± 5.73  3.86 ± 4.83^(a) 11.00 ± 9.63   2.30± 3.76^(a) O.Wi 3.77 ± 0.92  4.04 ± 0.91 3.66 ± 0.67 3.96 ± 0.83 3.94 ±1.13 3.76 ± 0.83 Rs.N/Ct.A 0.12 ± 0.17^(a) 0.21 ± 0.13 0.28 ± 0.18  0.12± 0.07^(a)  0.43 ± 0.26^(a) 0.19 ± 0.18 W.Wi 63.23 ± 13.61  68.63 ±15.09 61.36 ± 7.79  63.12 ± 17.35 65.28 ± 9.43  63.82 ± 8.31  omt 4.58 ±1.26^(a) 3.87 ± 1.04 3.76 ± 0.70 3.86 ± 0.74  6.45 ± 13.85 5.07 ± 2.65Po 1.32 ± 0.60^(a) 2.61 ± 1.40 4.65 ± 4.78 2.23 ± 1.60  6.78 ± 4.23^(a) 6.40 ± 4.22^(a) B.Ar 53.12 ± 5.50   52.82 ± 7.09  54.22 ± 5.97  54.94 ±6.55  55.81 ± 6.24  58.16 ± 8.79^(a) Ct.Ar 37.40 ± 3.75   35.35 ± 5.04 37.61 ± 3.86  38.10 ± 4.83  40.96 ± 4.30^(a) 40.83 ± 5.80^(a) Me.Ar15.72 ± 4.07   17.47 ± 4.22  16.61 ± 3.77  16.84 ± 3.74  14.85 ±4.72^(a) 17.34 ± 6.05 Abbreviations:OVX = ovariectomized;PTH1 = rhPTH(1-34) 1 μg/kg for 18 months;PTH1-W = withdrawal after treatment with rhPTH(1-34) 1 μg/kg for 12months;PTH5 = rhPTH(1-34) 5 μg/kg for 18 months;PTH5-W = withdrawal after treatment with rhPTH(1-34) 5 μg/kg for 12months.^(a)Statistically significant compared to OVX controls (p < 0.05).^(b)See table 4.2 for description of variables.^(c)Data are expressed as mean ± standard error of the mean (SEM).

The analysis by histomorphometry and polarized Fourier transforminfrared microscopy revealed that administration of PTH improved bonequality by replacing old bone (large crystallites) with young bone(range of sized crystallites, tending to smaller size). Further, uponwithdrawal of PTH from monkeys given low doses, there is an additionalbenefit as the matrix becomes optimally mineralized, and thecrystallites mature. The data derived from histomorphometry and Fouriertransform infrared microscopy show an unexpected benefit on bone qualityof the cortical bone as optimal mineralization occurs the mineral phasematures.,

3D Finite Element Modeling Studies

Examination of the middle 500 μm slice of L-5 showed a 21% increase inBMD for PTH compared to ovariectomized that was due to a 27% increase inBMC with no change in the cross sectional area. Analysis of the centrumfrom PTH showed a 73% increase in BV/TV that was due to a 30% increasein Tb.Th and 37% greater Tb.N, compared to ovariectomized.. Connectivityanalysis for this region showed a 140% greater node density (node/tissuevolume) and 286% greater node-to-node struts for the PTH vertebra.

Histogram distribution analysis of bone voxel densities for PTH showed adecrease in the proportion of low densities (0-355 mg/cc), an increasein middle densities (356-880 mg/cc), with little effect on the highdensity voxels (887-1200 mg/cc), compared to ovariectomized (FIG. 8).Most striking was the shift to a greater bone voxel density in thecortical bone compartment following withdrawal of treatment for 6 months(FIG. 8).

The proportion of vertebral bone elements (voxels) that fell within acertain range of BMD values was calculated. The BMD ranges chosen werethe following: low BMD, 0-300 mg/cc; medium BMD, 300-700 mg/cc; highBMD, 700-1000 mg/cc; and cortical BMD, >1000 mg/cc (Table 13). Comparedto ovariectomized controls, PTH treatment significantly decreased thevolume of low BMD bone and increased the volume of medium BMD bone.After withdrawal of PTH, there was a decrease in medium BMD bone and anincrease in high BMD bone, indicating that medium BMD bone became moredense. TABLE 13 Percentages of L5 vertebral volume grouped by BMD values(mean ± SEM) Treatment low BMD (%) medium BMD (%) high BMD (%) corticalBMD (%) ovariectomized 30.4 ± 2.2  48.7 ± 1.6 19.9 ± 1.2 0.9 ± 0.3 PTH17.7 ± 1.6*  58.9 ± 1.9* 22.8 ± 2.7 0.6 ± 0.3 PTH-W 22.4 ± 1.3* 49.7 ±1.2  27.0 ± 1.6* 0.8 ± 0.2

TABLE 14 BMC at the midlevel of the L5 vertebra and vertebral effectivestrain Treatment BMC (mg) effective strain (ustrain) ovariectomized 37.2± 1.6  701 ± 64  PTH 47.4 ± 1.5* 447 ± 36* PTH-W 44.2 ± 1.2* 539 ± 34**statistically different (p < 0.05 by Fisher's PLSD test)

The data summarized in FIG. 8 show that L-5 vertebra from cynomolgusmonkeys treated for 18 months with PTH respond with significantincreases in bone mass, trabecular thickness, and trabecularconnectivity, with marginal effects on the outer dimensions (X-Area) ofthe vertebra. Analysis of the distribution of bone elements in L-5showed that the heavily mineralized bone regions change the least withno evidence of bone sclerosis. Rather, it is the porous trabecular bonethat responded the most to PTH. The shift in BMD led to a substantialreduction in the axial strain, indicating mechanical improvement, Asclearly shown in the histograms of PTH and ovariectomy BMD, PTHconverted the low density bone voxels into medium density voxels with nosignificant effect on the high density voxels.

The data summarized in Table 2 show that the BMC through the middle ofthe vertebrae was significantly increased by PTH treatment and abeneficial effect of PTH remained after 6 months of withdrawal. Theaverage mechanical strain in the vertebra was reduced 36% by PTHtreatment and remained 23% below OVX after withdrawal of PTH. This studyindicates that withdrawal of PTH treatment for 6 months did not lead toresorption of newly formed bone, but instead there was a beneficialredistribution of medium density bone into lower and higher densitybone. This redistribution led to continued strain reduction in thevertebrae and thus improved mechanical function.

Discussion

This primate study indicates that PTH, given in the absence of othermedicines that might affect bone, benefits both cortical and trabecularbone to increase overall skeletal bone mass. Moreover, withdrawal of PTHdid not result in significant loss of benefits associated with PTHtreatment over a period of at least 2 remodeling cycles.

Surrogate markers have been used in other trials to indicate activity inbone, and they assumed that changes in value reflect changes in bonemass. Although there are published data on humans and primates to showthat both formation and resorption markers increase, consistent withactivation of bone turnover, for example, during early menopause or inactive disease states, the high turnover is considered to be indicativeof bone loss. In adolescence, high turnover during maturation of thehuman skeleton has been less well studied, but is accompanied by ananabolic gain in bone mass. Such a phenomenon would be totallyunexpected in drug therapy of osteoporosis, according to current art.Thus, the increase in bone turnover markers is inconsistent with theknown anabolic effect of PTH to increase bone mass and strength, asshown by data in the present study.

The data from this 18 month study on cynomolgus monkeys supports thefollowing unexpected findings:

-   -   Overall significant increase in total skeletal mass    -   Significant increase in bone mass and strength at the femur        neck.    -   No evidence of “steal” from cortical bone to increase trabecular        bone. Increase in bone mass and strength were statistically        significant at sites enriched for either cortical bone (femur        neck) or trabecular bone (lumbar vertebrae). At purely cortical        bone sites (femur mid-diaphysis), there was a trend for PTH to        stabilize or slightly increase bone mass and strength, compared        to ovariectomized controls.    -   Changes in bone markers in ovariectomized monkeys (and humans)        do not reflect the beneficial, anabolic effects of PTH on the        skeleton. Use of body fluids from primates in the present study        allows development of new and more valid surrogate markers.    -   Retention of the gain in bone mass and strength for at least 2        remodeling cycles after withdrawal of treatment.

This PTH primate study differs from published studies on rhesus andcynomolgus monkeys in that it used a large sample size to provideappropriate statistical power to detect differences that may not havebeen apparent in previous much smaller studies; controls included bothovariectomized primates (used in published studies) and sham operated,but intact primates. The latter control group has not been previouslyreported in this type of study, so that some of the benefits of PTH, andthe restoration of certain measures to sham control levels, compared tovalues for ovariectomized animals were assessed for the first time.

CONCLUSIONS

This 18-month study in mature, feral, ovariectomized (OVX) cynomolgusmonkeys, Macaca fascicularis, assured efficacy and safety in bonefollowing treatment with rhPTH(1-34) for either 12 months followed bywithdrawal of treatment for 6 months, or treatment for 18 months.rhPTH(1-34) significantly increased bone mass and strength of the spineand femur neck above ovariectomized controls to levels equivalent to orgreater than those of sham controls. In ovariectomized monkeys treatedwith rhPTH(1-34), measures of calcium homeostatis (serum calcium,phosphate, and 1,25-dihydroxyvitamin D) were restored to that of shamcontrols. Serum, urine, and histomorphometry measures used to assessbone turnover showed rhPTH(1-34) maintained formation rates equivalentto or higher than those of ovariectomized controls, while biochemicalmarkers of bone resorption remained equivalent to those of shamecontrols. In all animals treated with rhPTH(1-34) for up to 18 months,pharmacokinetic measures did not change with time, and there was noaccumulation of rhPTH(1-34). There was no evidence of sustainedhypercalcemia or kidney pathology after 18 months of treatment. Therewere no changes in mineralization or remodeling periods. The net gain inskeletal bone mineral content observed with rhPTH(1-34) may be explainedby increased bone formation rate and bone forming surface with little orno effect on bone resorption. There were significant increases in bonemineral content, bone mineral density and biomechanical measures ofstrength, including toughness and stiffness, at clinically relevantsites such as the spine, femur neck and proximal tibia.

rhPTH(1-34) increased the rate of turnover in cortical bone of midshaftof the humerus and radius, but did not significantly alter bone mass orbiomechanical measures of strength compared to either ovariectomized orsham controls. However, the increase in cortical width and/or corticalbone area is consistent with an increase in cross-sectional moment ofinertia, a measure of strength and stiffness. rhPTH(1-34) had nosignificant effects on the intrinsic material properties of corticalbone. Endocortical bone formation was stimulated, thus increasingcortical width and intracortical porosity. It appears that these changesin porosity are responsible for maintaining the elasticity of the bone.

In monkeys, 12 months of treatment with rhPTH(1-34) followed bywithdrawal for 6 months was associated with smaller, but stillsignificant, gains in bone mass and strength in the spine and femurneck. Following withdrawal, no significant effects were noted on thecortical bone midshaft of the humerus and radius. Bone markers andhistomorphometry showed trends to return to the low turnover valuesmeasured in sham controls.

In vivo mechanistic studies in rodents showed that genes associated withanabolic outcomes of rhPTH(1-34) are upregulated within 1 to 6 hours,and the increase in bone forming surfaces can be detected within 24hours after the first dose in the absence of detectable effects onresorption. rhPTH(1-34) appears to recruit osteoprogenitors in S-phase,and stimulate their differentiation into osteoblasts, thereby rapidlyincreasing the percent bone forming surfaces. Single or multipleinjections of rhPTH(1-34) may be given within a 1-hour period to inducethe anabolic effect in bone. However, when the equivalent dose is givenin young rats as multiple injections over 6 hours or 8 hours, theanabolic effect was abrogated, suggesting that brief, limited exposureto rhPTH(1-34) is required to induce the anabolic effect.

In summary, rhPTH(1-34) is anabolic on the bone in monkeys and rabbits,increasing bone mass and biomechanical strength measures at clinicallyrelevant sites such as the lumbar spine and femur neck by selectivestimulation of bone formation. The increases in bone turnover,endocortical surface formation, and porosity, detected byhistomorphometry at cortical sites, did not alter bone mass orbiomechanical measures of bone strength, but did increase the crosssectional moment of inertia by increasing cortical bone area and/orwidth,

These studies demonstrate that administration of parathyroid hormonereceptor activators, such as recombinant human PTH(1-34) improve bonequality both during and following treatment. In fact, administering PTHonce daily for 18 months, or at the same doses for 12 months followed bya 6 month withdrawal phase, showed marked improvement of the quality ofcortical bone of the humerus as analyzed by histomorphometry andpolarized Fourier transform infrared (FTIR) microscopy. This analysisrevealed that administration of PTH improved bone quality by replacingold bone (large crystallites) with young bone (range of sizedcrystallites, tending to smaller size). Thus, administration of PTH canincrease cortical bone quality, improve mineralization, and acceleratemineralization and replacement of old bone by new bone.

Further, upon withdrawal of PTH from monkeys given low doses, there isan additional benefit as the matrix becomes more optimally mineralized,and the crystallites mature. That is, at low doses, PTH can haveadditional benefits during the withdrawal phase of treatment byenhancing mineralization. These data indicate benefits of a finiteregime of treatment with PTH followed by a withdrawal period to achieveenhanced benefit. Current definitions of bone quality do not includethese aspects of improved mineralization.

In earlier studies of a treatment phase of PTH followed by a phase of notreatment, the treatment phase was less than 1 month. The prolonged butfinite treatment phase of 18-24 months followed by a period of at least2 remodeling cycles has not previously been explored. The continuedbenefit in primates after withdrawal of treatment is in marked contrastto results achieved in rodents upon dosing with PTH. Studies of ratshave uniformly shown that bone is rapidly lost following withdrawal oftreatment. Gunness-Hey, M. and Hock, J. M. (1989) Bone 10: 447-452;Shen, V. et al. (1993) J. Clin Invest 91: 2479-2487; Shen, V. et al.(1992) Calcif Tissue Int. 50: 214-220; and Mosekilde, L. et al. (1997)Bone 20: 429-437.

Such a method of enhancing bone mineralization has not previously beenobserved and is unexpected, revealing a new method by which PTHstrengthens and toughens bone and can prevent fractures. This new methodincludes enhancing and regulating mineralization, to provide tougher,stiffer, more fracture resistant bone. Such beneficial effects requiremore than new matrix formation. These findings indicate that PTH hasbenefits in patients with immobilized bones or skeletons, or inskeletons deficient in mineral, provided there is also adequate calciumand vitamin D supplementation.

Example 3

Increased Bone Strength and Density, and Reduced Fractures UponAdministration of rhPTH(1-34) to Humans

-   Number of Subjects:    -   rhPTH(1-34): 1093 enrolled, 848 finished.    -   Placebo: 544 enrolled, 447 finished.-   Diagnosis and Inclusion Criteria:    -   Women ages 30 to 85 years, postmenopausal for a minimum of 5        years, with a minimum of one moderate or two mild atraumatic        vertebral fractures.-   Dosage and Administration:    -   Test Product (blinded)    -   rhPTH(1-34): 20 μg/day, given subcutaneously    -   rhPTH(1-34): 40 μg/day, given subcutaneously    -   Reference Therapy (blinded)    -   Placebo study material for injection-   Duration of Treatment:    -   rhPTH(1-34): 17-23 months (excluding 6-month run-in phase)    -   Placebo: 17-23 months (excluding 6-month run-in phase)-   Criteria for Evaluation:

Spine x-ray; serum biological markers (calcium, bone-specific alkalinephosphatase, procollagen I carboxy-terminal propeptide); urine markers(calcium, N-telopeptide, free deoxypyridinoline); 1,25-dihydroxyvitaminD; bone mineral density: spine, hip, wrist, and total body; height;population pharmacokinetic; bone biopsy (selected study sites). PlaceboPTH-20 PTH-40 (N = 544) (N = 541) (N = 552) p-value Caucasian 98.9%98.9% 98.4% 0.672 Age 69.0 ± 7.0 69.5 ± 7.1 69.9 ± 6.8 0.099 Years postmenopausal 20.9 ± 8.5 21.5 ± 8.7 21.8 ± 8.2 0.273 Hysterectomized 23.8%23.1% 21.6% 0.682 Uterus + 0 or 1 ovary 57 51 58 Uterus + 2 ovaries 6157 51 Unknown 11 17 10 Previous osteoporosis drug 14.9% 15.5% 13.0%0.479 use Baseline spine BMD 0.82 ± 0.17  0.82 ± 0.17 0.82 ± 0.17 >0.990Baseline # of vert. fx >0.990  0 54 (10.4%) 45 (8.8%) 54 (10.1%)  1 144(27.8%) 159 (31.1%) 16 (31.6%)  2 128 (24.7%) 128 (25.0%) 125 (23.4%)  375 (14.5%) 67 (13.1%) 81 (15.1%)  4 59 (11.4%) 49 (9.6%) 45 (8.4%)  5 28(5.4%) 31 (6.1%) 21 (3.9%)  6 13 (2.5%) 20 (3.9%) 25 (4.7%)  7 6 (1.2%)7 (1.4%) 10 (1.9%)  8 9 (1.7%) 5 (1.0%) 3 (0.6%)  9 1 (0.2%) 0 2 (0.4%)10 1 (0.2%) 1 (0.2%) 0 Unspecified 26 29 17Results

Data from this clinical trial including a total of 1637 women treatedwith recombinant human parathyroid hormone (1-34), rhPTH(1-34)0, 20, or40 μg/kg/day, and supplemented with vitamin D and calcium, for 18-24months, showed results reported in Tables 15-19.

Table 15 illustrates data showing the reduction upon treatment with PTHof the number and severity of vertebral fractures. Comparing all PTHtreated patients with placebo, the overall reduction in number ofpatients with vertebral fractures was 67% (p<0.001), with a 65%reduction (p<0.001) at 20 μg/day PTH compared to placebo, and a 69%reduction at 40 μg/day PTH compared to placebo (Table 15). Comparing allPTH treated patients with placebo, the overall reduction in number ofpatients with multiple vertebral fractures was 81% (p<0.001), with a 77%reduction (p<0.00l) at 20 μg/day PTH compared to placebo, and a 86%reduction at 40 μg/day PTH compared to placebo. Comparing -all PTHtreated patients with placebo, the overall reduction in number ofpatients with moderate to severe vertebral fractures was 84% (p<0.001),with a 90% reduction (p<0.001) at 20 μg/day PTH compared to placebo, anda 78% reduction at 40 μg/day PTH compared to placebo (Table 15). TABLE15 Effect of treatment with PTH on number and severity of vertebralfractures. Placebo 20 μg/day PTH 40 μg/day PTH (n* = 448) (n = 444) (n =434) Number and 64 22  19  percentage of (14.3%)  (5.0%) (4.4%) patientswith new vertebral fractures Number and 22 5 3 percentage of (4.9%)(1.1%) (0.7%) patients with 2 or more new vertebral fractures Number and42 4 9 percentage of (9.4%) (0.9%) (2.1%) patients with new moderate tosevere fractures***n = number of patients with both baseline and endpoint x-rays**Moderate fracture results in more than 25% loss of vertebral height(or an equivalent measure). Severe fracture results in more than 40%loss of vertebral height (or an equivalent measure). Fractures are asdefined by Genant et al. (1993) Vertebral fracture assessment using asemiquantitative technique; J. Bone & Min Res 81137-1148.

Table 16 illustrates the effect of treatment with PTH on the number offractures at various non-vertebral bones throughout the body. The numberof fractures apparently decreased at each of the hip, radius, ankle,humerus, ribs, foot, pelvis, and other sites (Table 16). The reductionis statistically significant when viewed as the reduction in the totalnumber of fractures among the PTH treated patients compared to theplacebo treated patients. The reduction is even more significant whenconsidered as the reduction in the total number of fractures of hip,radius, ankle, humerus, ribs, foot, and pelvis among the PTH treatedpatients compared lo the placebo treated patients (Table 16). TABLE 16Effect of treatment with PTH on number of non-vertebral fractures.Placebo PTH-20 PTH-40 p-values (N = 544) (N = 541) (N = 552) OverallPTH-pbo* 20-pbo 40-pbo Hip 4 2 3 0.718 0.474 0.417 0.690 Radius 13 7 100.404 0.236 0.180 0.504 Ankle 4 2 2 0.601 0.313 0.417 0.403 Humerus 5 43 0.767 0.534 0.744 0.465 Ribs 10 5 5 0.277 0.109 0.197 0.184 Foot 4 1 40.374 0.474 0.151 0.983 Pelvis 3 1 0 0.171 0.076 0.319 0.081 Other 16 149 0.338 0.296 0.723 0.146 Total 53 34 32 0.024 0.007 0.036 0.015 Total41 21 24 0.013 0.003 0.010 0.025 w/o “Other”*Placebo (pbo)

The effect of PTH on bone mineral content (BMC), bone mineral density(BMD), and bone area were determined by dual energy absorptiometry(DEXA), and the results are reported in Tables 17-19. PTH administrationcaused apparent increases in BMC at the patient's lumbar spine, femurand hip, wrist, and throughout the patient's whole body (Table 17).Treatment with PTH caused significant increases in the patient's BMD atthe lumbar spine, femur and hip (Table 18). The increases at the lumbarspine, femur and hip were statistically significant with p<0.001 (Table18). Bone area apparently increased upon PTH treatment for the patient'slumbar spine, femur and hip (Table 19). The increases were statisticallysignificant for the lumbar spine and hip neck (Table 19).

The effect of PTH on the whole body the measure of bone quantity andquality, BMC, is particularly significant. This whole body effectindicates that the amount of bone in the patient's body is increasing.PTH does not merely result in moving bone mass from one portion of thepatient's body to another. Instead, treatment with PTH increases theamount and quality of bone in the patient's body.

FIGS. 9 and 10 illustrate the increases over time in lumbar spine BMDand femur/hip neck BMD, respectively, for PTH treated and placebocontrol patients. The patient's lumbar spine BMD increases steadily forat least about 18 months, with no or a less significant increase overthe subsequent months. The patient's femur/hip BMD apparently increasesfor at least 18 months, and may increase upon further duration of PTHtreatment. TABLE 17 Effect of PTH on bone mineral content expressed asendpoint % change (SD) from baseline p- Placebo PTH-20 PTH-40 valueLumbar 1.60 (6.92) 11.85 (8.83)  16.62 (11.1)  <0.001 spine Femur/HipTotal −0.38 (5.18) 3.50 (6.26) 4.78 (6.70) <0.001 Neck −0.51 (7.06) 2.99(7.26) 5.80 (8.71) <0.001 Trochanter  0.98 (14.97)  5.68 (15.58)  6.53(15.33) <0.001 Intertro- −0.23 (6.28) 3.59 (7.32) 4.99 (7.79) <0.001chanter Ward's  0.01 (14.75)  5.36 (14.78)  8.86 (17.02) <0.001 triangleWrist Ultra- −1.67 (7.44) −0.25 (6.53)  −1.88 (7.97) 0.184 distal ⅓−1.19 (6.12) −1.37 (4.51)  −3.04 (6.09) 0.025 radius Whole body −0.74(4.76) 1.30 (4.48)  2.28 (5.44) <0.001

TABLE 18 Effect of PTH on bone mineral density expressed as endpoint %change (SD) from baseline p- Placebo PTH-20 PTH-40 value Lumbar  1.13(5.47) 9.70 (7.41) 13.7 (9.69) <0.001 spine Femur/Hip Total −1.01 (4.25)2.58 (4.88) 3.60 (5.42) <0.001 Neck −0.69 (5.39) 2.79 (5.72) 5.06 (6.73)<0.001 Trochanter −0.21 (6.30) 3.50 (6.81) 4.40 (7.45) <0.001 Intertro-−1.29 (4.53) 2.62 (5.52) 3.98 (5.96) <0.001 chanter Ward's  −0.80(11.73)  4.19 (11.93)  7.85 (13.24) <0.001 triangle Wrist Ultra- −1.89(7.98) −0.05 (7.14) −1.76 (7.20) 0.108 distal ⅓ −1.22 (3.37) −1.94(4.07) −3.17 (4.62) 0.001 radius

TABLE 19 Effect of PTH on bone area expressed as endpoint % change (SD)from baseline p- Placebo PTH-20 PTH-40 value Lumbar 0.46 (2.97) 2.52(3.52) 3.34 (3.72) <0.001 spine Femur/Hip Total 0.54 (3.02) 0.84 (3.16)1.05 (2.98) 0.144 Neck 0.04 (4.60) 0.27 (4.91) 0.81 (5.56) 0.035Trochanter  0.95 (12.75)  1.99 (12.16)  1.92 (11.30) 0.197 Intertro-1.01 (5.17) 1.01 (4.99) 1.01 (4.89) 0.964 chanter Ward's 0.44 (7.60)1.13 (7.34) 0.99 (8.06) 0.309 triangle Wrist Ultra- 0.25 (6.40) −0.25(6.00)  −0.39 (4.80)  0.653 distal ⅓ −0.02 (5.73)  0.52 (3.40) 0.01(4.42) 0.586 radius

In summary, the data presented above indicate that patients treated withPTH have reduced fractures. Specifically, PTH treatment reduced by morethan 66% the number of patients with prior vertebral fractures whosuffered new vertebral fractures. PTH treatment also reduced by morethan 78% the number of patients with prior vertebral fractures whosuffered new, multiple vertebral fractures. In addition, PTH decreasedthe severity of vertebral fractures, with a significant reduction by 78%in the number of patients with moderate or severe fractures. Patientsreceiving PTH benefited from a significant reduction in allnon-vertebral fractures (including fractures of hip, radius, wrist,pelvis, foot, humerus, ribs or ankle) with significance at a level ofp<0.007. Bone quality increases as well. Patients with prior fracturebenefited from a significant increase in bone mineral content of thehip, spine and total body. This increase indicates that fracturereduction at these sites can occur as early as after 12 months oftherapy.

DISCUSSION

These data on fractures are the first data on fracture reduction by PTHin humans. These findings demonstrate an improvement in bone quality andbone strength, like the preclinical data reported hereinabove. Theseresults also show benefits in bone quality and strength at non-vertebralsites. The findings of a reduction in the numbers of fractures sustainedduring the 18-23 month period of treatment has not previously beenobserved in clinical or preclinical studies.

The question of whether PTH alone increases toughness and strength ofbone to improve resistance to fracture has not previously been tested inhumans. The published literature has consistently suggested that PTHmust be given in combination with an anti-resorptive or estrogen.Previous, published clinical trials included patient populations toosmall to determine a significant reduction of fracture. In one study thebenefits of PTH alone could not be assessed because there were noplacebo controls. In a second study, employing the commonly accepteddefinition of fracture, no reduction in fracture was observed.

The findings of a reduction of fractures at combined non-vertebral sitesis particularly unexpected in light of the common belief that PTH hasnegative effects at such sites. Common dogma holds that PTH willincrease cortical porosity and therefore weaken bone, especially earlyin therapy. Further, this dogma asserts that cortical bone sites are athigh risk of fracture and that PTH will offer no benefit in fracturereduction at non-vertebral sites. The dogma also holds that PTH alone isunlikely to be efficacious and will require concurrent anti-resorptivetherapy to block negative effects on cortical bone. The present datademonstrate the previously unobserved benefits of PTH given to patientsreceiving vitamin D and calcium supplements. Unexpectedly, PTHstrengthens bone to reduce the number of new fractures in a patient atrisk for multiple fractures of the spine, at risk for additionalnon-vertebral fractures, at risk for moderate to severe additionalfractures of the spine, and the like.

This clinical study on post-menopausal women showed particular benefitsfrom treating patients with low dose (20 μg/day) since the dose of PTH(which, at high doses, could show side effects in some patients) wasreduced, but fracture prevention and fracture reduction was retained,and similar to those noted at the high dose (40 μg/day). The FT-IRmonkey data provide a possible, but not limiting, mechanisticexplanation. The monkey study shows that low dose PTH increased crystalformation and accelerated mineralization in cortical bone. In addition,low dose monkeys showed additional benefits after withdrawal, as PTHenhanced mineral content of the bone. The present data demonstrate thenovel finding that PTH given at low doses to patients receiving vitaminD and calcium supplements, is effective in preventing both vertebral andnon-vertebral fractures. Contrary to popular belief, PTH strengthensbone at non-vertebral sites to prevent new fractures or reduce theseverity of fractures, apparently by improving the mineralization andmineral content of the bone.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention. All publications andpatent applications in this specification lo are indicative of the levelof ordinary skill in the art to which this invention pertains.

1. A method for increasing toughness or stiffness of bone at a site of apotential or actual trauma in a subject in need thereof, comprisingadministering to the subject an effective amount of a parathyroidhormone.
 2. The method of claim 1, wherein the trauma is a potentialtrauma comprising a fracture, a surgery, or an orthopedic procedurecomprising manipulation of a bone at a site of abnormally low bone massor poor bone structure.
 3. The method of claim 2, wherein the surgery isa joint replacement, a spine bracing, or a combination thereof.
 4. Themethod of claim 3, wherein the joint replacement comprises hipreplacement.
 5. The method of claim 2, wherein the fracture comprises avertebral fracture, a non-vertebral fracture, or a combination thereof.6. The method of claim 6, wherein the non-vertebral fracture comprises ahip fracture, a fracture of a distal forearm, a fracture of a proximalhumerus, a fracture of a wrist, a fracture of a radius, a fracture of anankle, a fracture of an humerus, a fracture of a rib, a fracture of afoot, a fracture of a pelvis, or a combination thereof.
 7. The method ofclaim 1, wherein the trauma is a potential trauma comprising traumarelated to hypoparathyroidism or to progression of kyphosis.
 8. Themethod of claim 1, wherein the trauma is an actual trauma comprising afracture.
 9. The method of claim 8, wherein the fracture comprises avertebral fracture, a non-vertebral fracture, or a combination thereof.10. The method of claim 9, wherein the non-vertebral fracture comprisesa hip fracture, a fracture of a distal forearm, a fracture of a proximalhumerus, a fracture of a wrist, a fracture of a radius, a fracture of anankle, a fracture of an humerus, a fracture of a rib, a fracture of afoot, a fracture of a pelvis, or a combination thereof.
 11. The methodof claim 1, wherein the bone comprises an immobilized bone or skeleton,a bone or skeleton deficient in mineral, or a combination thereof. 12.The method of claim 1, wherein the bone comprises cortical bone,cancellous bone, trabecular bone, or a combination thereof.
 13. Themethod of claim 12, wherein the bone comprises a site of attachment fora ligament, a tendon, a muscle, or a combination thereof.
 14. The methodof claim 1, wherein the trauma site is a hip, a spine, or a combinationthereof.
 15. The method of claim 14, wherein the trauma site comprises afemur neck, a trochantera of a femur, an ilium, or a combinationthereof.
 16. The method of claim 15, wherein the trauma site comprisescancellous bone of the ilium.
 17. The method of claim 14, wherein thetrauma site comprises a mid-thoracic vertebra, an upper lumbar vertebra,or a combination thereof.
 18. The method of claim 1, wherein the subjectis a woman at risk for osteoporosis.
 19. The method of claim 18, whereinthe subject is a postmenopausal woman.
 20. The method of claim 19,wherein the woman is independent of hormone replacement therapy or anantiresorptive.
 21. The method of claim 1, wherein the subject is awoman in an early stage of osteoporosis or in an advanced stage ofosteoporosis.
 22. The method of claim 1, wherein increasing toughness orstiffness comprises increasing toughness and stiffness.
 23. The methodof claim 1, wherein increasing toughness or stiffness comprisesdecreasing risk or probability of fracture.
 24. The method of claim 1,wherein increasing toughness or stiffness comprises increasingactivation frequency or bone formation rate in cortical and trabecularbone.
 25. The method of claim 1, wherein increasing toughness orstiffness comprises increasing bone mineral content, increasing bonemineral density, increasing trabecular number, increasing trabecularthickness, reducing marrow space, increasing trabecular connectivity,increasing connectivity, increasing resistance to loading, increasingperiosteal and endocortical bone formation, increasing corticalporosity, increasing cross sectional bone area and bone mass, increasingwork to failure, decreasing elastic modulus, or a combination thereof.26. The method of claim 1, wherein administering comprises subcutaneousadministration.
 27. The method of claim 1, wherein the parathyroidhormone is administered cyclically or intermittently.
 28. The method ofclaim 27, wherein cyclic administration comprises administering theparathyroid hormone for at least 2 remodeling cycles and withdrawingparathyroid hormone for at least 1 remodeling cycle.
 29. The method ofclaim 27, wherein cyclic administration comprises administering theparathyroid hormone for at least about 12 to about 24 months andwithdrawing parathyroid hormone for at least 6 months.
 30. The method ofclaim 1, wherein the parathyroid hormone is a fragmented hormoneselected from the group consisting of PTH(1-31), PTH(1-34), PTH(1-37),PTH(1-38), and PTH(1-41).
 31. The method of claim 1, wherein theparathyroid hormone is human PTH(1-34).
 32. The method of claim 1,wherein the parathyroid hormone is human PTH(1-84).
 33. The method ofclaim 1, wherein the parathyroid hormone is administered at a dose of atleast about 5 μg/kg/day.
 34. The method of claim 33, wherein the dose isabout 10 to about 40 μg/kg/day.
 35. The method of claim 1, furthercomprising administering calcium, vitamin D, or a combination thereof.36. The method of claim 1, wherein increasing toughness or stiffnesscomprises increasing bone mineral content of medium density bone. 37.The method of claim 1, wherein increasing toughness or stiffnesscomprises increasing bone mineral content of low and high density boneand reduction of bone mineral content of medium density bone.
 38. Themethod of claim 1, wherein increasing toughness or stiffness comprisesincreasing bone mineral content of medium density bone followed byincreasing bone mineral content of low and high density bone andreduction of bone mineral content of medium density bone.
 39. The methodof claim 1, wherein increasing toughness or stiffness comprises reducingthe size of crystallites in the bone.
 40. The method of claim 39,further comprising maturing crystallites of the bone.
 41. The method ofclaim 1, wherein increasing toughness or stiffness comprises increasingmineralization of the bone.
 42. The method of claim 1, whereinincreasing toughness or stiffness comprises reducing incidence offracture.
 43. The method of claim 42, wherein increasing toughness orstiffness comprises reducing incidence of vertebral fracture, reducingincidence of severe fracture, reducing incidence of moderate fracture,reducing incidence of non-vertebral fracture, reducing incidence ofmultiple fracture, or a combination thereof.
 44. A method for reducingthe risk of bone fracture in a subject in need thereof, comprisingadministering to the subject an effective amount of a parathyroidhormone.
 45. The method of claim 44, wherein the bone comprises a hip, aradius, an ankle, an humerus, a rib, a foot, a pelvis, a spine or acombination thereof.
 46. The method of claim 44, wherein the parathyroidhormone is a fragmented hormone selected from the group consisting ofPTH(1-31), PTH(1-34), PTH(1-37), PTH(1-38), and PTH(1-41).
 47. Themethod of claim 44, wherein the fracture comprises a vertebral fracture,a non-vertebral fracture, or a combination thereof.
 48. The method ofclaim 47, wherein the non-vertebral fracture comprises a hip fracture, afracture of a distal forearm, a fracture of a proximal humerus, afracture of a wrist, a fracture of a radius, a fracture of an ankle, afracture of an humerus, a fracture of a rib, a fracture of a foot, afracture of a pelvis, or a combination thereof.
 49. A process formanufacturing a medicament used for increasing toughness or stiffness ofbone at a site of potential or actual trauma, comprising combining aparathyroid hormone with a pharmaceutically acceptable carrier.
 50. Theprocess of claim 49, wherein the medicament comprises a stabilizedformulation of a parathyroid hormone.
 51. The process of claim 50,wherein the stabilized formulation comprises: a therapeuticallyeffective amount of parathyroid hormone; a polyol, such as mannitol orpropylene glycol; a buffering agent suitable for maintaining the pH ofthe composition within a range of about 3-7, such as an acetate ortartrate source; and water.
 52. The use of a parathyroid hormone in themanufacture of a medicament for reducing the risk of bone fracture in asubject in need thereof.
 53. The method of claim 52, wherein the bonecomprises a hip, a radius, an ankle, an humerus, a rib, a foot, apelvis, a spine or a combination thereof.
 54. The method of claim 52,wherein the parathyroid hormone is a fragmented hormone selected fromthe group consisting of PTH(1-3 1), PTH(1-34), PTH(1-37), PTH(1-38), andPTH(1-41).
 55. The method of claim 52, wherein the fracture comprises avertebral fracture, a non-vertebral fracture, or a combination thereof.56. The method of claim 55, wherein the non-vertebral fracture comprisesa hip fracture, a fracture of a distal forearm, a fracture of a proximalhumerus, a fracture of a wrist, a fracture of a radius, a fracture of anankle, a fracture of an humerus, a fracture of a rib, a fracture of afoot, a fracture of a pelvis, or a combination thereof.
 57. The use of aparathyroid hormone for preparing a composition used for reducing therisk of bone fracture in a subject in need thereof.
 58. The use of aparathyroid hormone in the manufacture of a medicament for increasingtoughness or stiffness of bone at a site of potential or actual trauma.59. The use of a parathyroid hormone for preparing a composition usedfor increasing toughness or stiffness of bone at a site of potential oractual trauma.